1 Microbiological Testing by Industry of Ready-to-Eat Foods Under FDA’s Jurisdiction for Pathogens 

2 (or Appropriate Indicator Organisms): Verification of Preventive Controls1 

3 ADOPTED 22 APRIL 2021, WASHINGTON, DC 

4 NATIONAL ADVISORY COMMITTEE ON MICROBIOLOGICAL CRITERIA FOR FOODS 

5 

6 NACMCF Executive Secretariat*, U.S. Department of Agriculture, Food Safety 

7 and Inspection Service, Office of Public Health Science, Stop 3777, PP3, 9-210B, 1400 Independence 

8 Avenue S.W., Washington, DC 20250-3700, USA 

9 

10 *Author for correspondence: Author for correspondence. Tel: 202-690-6537; Fax: 202-690-6364; E-mail: 

11 evelyne.mbandi@fsis.usda.gov. 

12 

13 Key words: Preventive controls, microbiological testing, verification testing, indicator organisms, 

14 environmental monitoring 

1 Participating agencies include the U. S. Department of Agriculture, Food Safety and Inspection Service; 

U.S. Department of Health and Human Services, Food and Drug Administration, and Centers for Disease 

Control and Prevention; U.S. Department of Commerce, National Marine Fisheries Service; and U.S. 

Department of Defense, Veterinary Service Activity. This article may be reproduced without prior 

permission. 

Disclaimer: Mention of trade names or commercial products in this publication is solely for the purpose 

of providing specific information and does not imply recommendation or endorsement by the U. S. 

Department of Agriculture and other participating agencies. 
 

 

 

 

 

 

 

 

 

 

 

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EXECUTIVE SUMMARY 

FDA’s final rule “Current Good Manufacturing Practice, Hazard Analysis, and Risk-Based 

Preventive Controls for Human Food” (the CGMP & PC rule) (46) requires a facility that has identified 

hazards requiring preventive controls to verify that the preventive controls are consistently implemented 

and are effectively and significantly minimizing or preventing the hazard. Verification activities for 

preventive controls for microbial hazards include, as appropriate to the facility, the food, and the nature 

of the preventive control and its role in the facility's food safety system, product testing for a pathogen 

(or appropriate indicator organism). FDA is seeking advice from the National Advisory Committee on 

Microbiological Criteria for Foods (NACMCF) on 1) the utility and necessity of industry testing certain 

ready-to-eat (RTE) foods for pathogens and 2) criteria industry could apply in determining what, if any, 

microbiological testing is appropriate for verifying pathogen control for the RTE foods produced in a 

facility. As these are FDA inquiries, the scope of NACMCF’s advice includes responses for dairy products, 

grain-based products, meals and entrees, nuts and nut/seed products, fruits and vegetables, and spices 

and herbs. 

The intent of this document is to provide examples and advice for manufacturers/processors to 

establish their own microbial targets and limits to meet preventive control requirements. It offers 

guidance for using microbiological testing for pathogens (or appropriate indicator organisms) to verify 

process control for pathogens in RTE foods under FDA’s jurisdiction. Advise provided by NACMCF is 

intended to guide decisions to be made by each firm based on their facility, ingredients used, processing, 

packaging, level of anticipated control, shelf life of the product, intended use, or potential storage and 

handling at retail or by the consumer. The NACMCF was specifically charged with offering guidance on: 1) 

principles and criteria a company should apply in determining the need for and in designing an effective 

microbial testing program to verify that processes are effectively controlling microbial pathogens; 2) 

situations in which testing other than for pathogens or indicator organisms would be an appropriate 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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verification activity for a company; 3) situations where verification testing by a company would not be 

necessary if there is evidence that the appropriate treatment was, in fact, applied; 4) when microbial 

testing is an appropriate verification activity, considerations a company should apply in selecting the test 

microorganisms and what are appropriate indicator microorganisms for verifying processes that 

adequately control pathogens; 5) principles and criteria a company should apply in determining the 

frequency of testing finished product to determine if the company’s food safety system for that product 

is effective; 6) situations in which testing at sites other than at the end of the process can achieve the goal 

of verifying the adequacy of control of microbial hazards; 7) the impacts of environmental monitoring on 

frequency and extent of product testing verification activities by companies; and 8) criteria and action a 

company should apply in determining that microbial testing results indicate a loss of process control and 

to what extent should verification testing be increased, how far upstream and downstream should it go, 

and when and how should it be scaled back. 

BACKGROUND 

In 2015, FDA published its final rule “Current Good Manufacturing Practice, Hazard Analysis, and 

Risk-Based Preventive Controls for Human Food” (the CGMP & PC rule) in title 21 of the Code of Federal 

Regulations (CFR) part 117 (51). A facility that has identified hazards requiring preventive controls must 

verify that the preventive controls are consistently implemented and are effectively and significantly 

minimizing or preventing the hazard. As specified in 21 CFR 117.165, verification activities for preventive 

controls for microbial hazards include, as appropriate to the facility, the food, and the nature of the 

preventive control and its role in the facility's food safety system, product testing for a pathogen (or 

appropriate indicator organism). FDA has indicated that such product testing is a verification activity to 

help assess and verify the effectiveness of a food safety plan and the facility’s capability to consistently 

deliver against it, not to establish the acceptability of every lot or batch. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Because of the flexibility FDA provided in the rule, advice from NACMCF on 1) the utility and 

necessity of industry testing ready-to-eat (RTE) foods for pathogens and 2) criteria industry could apply in 

determining what, if any, microbiological testing is appropriate for verifying pathogen control for the RTE 

foods produced in a facility, would be highly beneficial for industry. Such advice should include the test 

microorganism(s), the sampling plan that should be used, the type of test (e.g., presence/absence or 

enumeration), the frequency of such testing, interpretation of results, and actions to take when such 

testing indicates a loss of control. Advice from NACMCF should address the appropriate use of enzymatic 

indicators that heat-based processes have been applied (e.g., alkaline phosphatase for pasteurization of 

milk) and whether there are situations where verification testing of products by industry would not be 

necessary if there is evidence that the appropriate treatment was applied. 

A 2013-2015 NACMCF Subcommittee addressed a charge from the Department of Defense (DoD) 

on Microbiological Criteria as Indicators of Process Control or Insanitary Conditions (35). That charge was 

to develop microbiological and other possible criteria for DoD auditors to better evaluate process control 

and insanitary conditions at the point of production. Some of the information developed in the final report 

of that Subcommittee (35) were considered in addressing this charge. However, the focus here is on 

practical advice for manufacturers/processors subject to the preventive control requirements in 21 CFR 

part 117 about when they should use microbiological testing for pathogens (or appropriate indicator 

organisms) to verify process control for pathogens in RTE foods under FDA’s jurisdiction. For this 

document, process control refers to the entire operation (e.g., entire food safety system/process). It is 

not restricted to process preventive controls. 

A food safety system and the manufacturing process managed by that system are in control when, 

within the limits of a stable and predictable process variation, all food safety hazards are controlled to an 

acceptable level (29). 
 

 

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FOOD CATEGORIES OF CONCERN 

Dairy Products 

Butter, margarine 

Cheese, hard (e.g., Cheddars), extra hard, grating (e.g., Parmesan, Romano) 

Cheese, fresh (Queso fresco), soft, soft-ripened (Camembert), semi-soft (Edam, Gouda), veined 

cheeses (Roquefort, Gorgonzola) 

Cultured, pH < 4.8 

Cultured, pH > 4.8 and <5.4 

Dried products (including dairy ingredients used to make infant formula) 

Frozen desserts 

Milk and milk products (fluid) 

Grain-Based Products 

RTE baked items, refrigerated or time-temperature controlled for safety (TCS) 

RTE baked items, shelf stable or non-TCS 

RTE cereals 

RTE cold-pressed bars 

Meals and Entrees 

RTE deli salads 

RTE sandwiches 

“Heat and eat” meals/entrees 

Nuts (including tree nuts and peanuts) and Nut/Seed Products 

RTE nuts not processed for lethality (e.g., chopped untreated tree nuts) 

RTE nuts processed for lethality (e.g., roasted tree nuts, almond milk, coconut milk) 

RTE nut/seed butters processed for lethality (e.g., peanut butter, sunflower butter) 
 

 

 

 

 

 

 

 

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Fruits and Vegetables 

RTE fresh-cut fruits (e.g., cut melon, sectioned grapefruit, sliced pineapple) 

RTE fresh-cut vegetables (e.g., cut celery stalks, peeled baby carrots, sliced mushrooms, shredded 

cabbage, chopped lettuce) 

RTE dried/dehydrated fruits (e.g., dried cranberries, raisins, dried apricots) 

Packaged uncut leafy greens (e.g., spinach leaves, baby greens leaves) 

Spices and Herbs (include consideration for intrinsic properties in certain spices and herbs (e.g., cinnamon, 

cloves, oregano) that can interfere with test methodology and risk from added components in 

spice blends) 

RTE spices and spice blends, not processed for lethality 

RTE spices and spice blends, processed for lethality 

Dried, chopped herbs 

CHARGE QUESTIONS TO THE COMMITTEE 

1. For the food categories listed above, what principles and criteria should a company apply in 

determining the need for and in designing an effective microbial testing program to verify that processes 

are effectively controlling microbial pathogens? 

2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

enzymes, would be an appropriate verification activity for a company? 

3. Are there situations where verification testing by a company would not be necessary if there is 

evidence that the appropriate treatment was, in fact, applied? 

4. When microbial testing is an appropriate verification activity, what considerations should a 

company apply in selecting the test microorganism (e.g., specific pathogen or specific indicator organism) 
 

 

 

 

 

 

 

 

 

 

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and type of test (e.g., presence/absence or enumeration)? What are appropriate indicator 

microorganisms for verifying processes that adequately control pathogens? 

5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

6. Generally microbial testing by a company to verify process control is conducted on “finished 

product.” Are there situations in which testing at sites other than at the end of the process can achieve 

the goal of verifying the adequacy of control of microbial hazards? Describe the situations and the testing 

that would be appropriate. 

7. The CGMP & PC rule requires environmental monitoring for an environmental pathogen (e.g., 

Listeria monocytogenes, Salmonella) or for an appropriate indicator organism as a verification activity if 

contamination of an RTE food with an environmental pathogen is a hazard requiring a preventive control 

(such as sanitation controls). What impact does environmental monitoring have on frequency and extent 

of product testing verification activities by companies? Note: Committee changed “should” to “does” for 

responding to this charge. 

8. What criteria should a company apply in determining that microbial testing results indicate a 

loss of process control? What actions should a company take if test results indicate a loss of process 

control? When verification testing indicates loss of process control, to what extent should verification 

testing be increased, how far upstream and downstream should it go, and when and how should it be 

scaled back? 

COMMITTEE’S APPROACH TO ANSWERING THE CHARGE 

The Committee leveraged the expertise of the Committee members, additional experts, published 

literature and government documents to develop guidance for firms considering product testing (in 

process or finished product) as an activity to verify that their pathogen controls are effective. In addition 
 

 

 

 

 

 

 

 

 

 

 

 

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to answering charge questions, appendices were developed for each food grouping as examples of 

considerations in choosing type and frequency of microbial testing. With rare exceptions noted in the 

tables within each appendix, microbial targets and limits are not for lot disposition. Rather, the examples 

provide reference points for expected microbial population limits in foods that are produced with good 

quality ingredients, validated lethality steps or other process controls, and rigorous sanitation and 

environmental monitoring programs. Each firm should establish their own microbial targets and limits 

depending on the facility, ingredients used, processing, packaging, level of anticipated control, shelf life 

of the product, intended use, or potential storage and handling at retail or by the consumer. 

INTRODUCTION 

Historically, the role of HACCP was to effectively control hazards such as microbial contamination 

and if properly implemented, would reduce the need for finished product testing for pathogens. But, 

while this concept works to reduce or eliminate pathogen testing for some foods, other food products still 

rely on frequent finished product testing for pathogens, whereas other foods focus on testing for indicator 

organisms to ensure process control. 

Each individual firm should consider if microbial testing of product is an appropriate verification 

activity, and if so, what are the target microorganisms that are appropriate for a given commodity? Should 

pathogens or indicators organisms be tested, or both? What is the role of environmental monitoring, and 

can it be sufficient? 

Microbial testing results can serve as an early warning that the process is drifting out of control 

or signal potential catastrophic failures. Data collected (e.g., enumeration of indicator organisms, positive 

environmental tests) should be analyzed on an ongoing basis for trends, be used to develop statistical 

process control, modify microbial limits as appropriate, and establish responses to results that exceed 

those limits. 
 

 

 

 

 

 

 

 

 

 

 

 

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RESPONSES 

Charge Question 1. For the food categories listed above, what principles and criteria should a 

company apply in determining the need for and in designing an effective microbial testing program to 

verify that processes are effectively controlling microbial pathogens? 

Microbiological testing of in-process or finished product is appropriate for some, but not all, 

ready-to-eat (RTE) foods to verify preventive controls in a Food Safety Plan. While finished product 

testing is generally not effective for controlling food safety, testing can be used for process and product 

verification (30, 55). Product testing could be used to verify that the overall production continuum is in 

control as the final product reflects the adequacy of the processing system controls and the processing 

environment. In addition, finished product testing can be useful in detecting catastrophic failures. A food 

processing facility can apply several criteria to determine whether microbiological testing is appropriate 

for in-process or RTE finished products. The following eight questions were used to determine the 

conditions that determine if microbiological testing is appropriate for each commodity group and their 

example foods. A comparison of answers to each question for the various commodities are given in Table 

1. Detailed answers to questions for each commodity are provided in Appendices A-F. Criteria to consider 

include: 

1. Have pathogens been associated with the food or its ingredients and has the food been 

associated with foodborne illness? All of the raw commodities (i.e., those without a lethality step) 

discussed in this document have been associated with pathogens and/or foodborne illness. Such 

pathogens include Salmonella, Shiga toxin-producing Escherichia coli (STEC), Campylobacter, Listeria 

monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, and Clostridium 

botulinum. Depending on the processing environment and food, a frequent concern is post-lethality 

contamination. Foodborne illness can result from long-term survival of low infectious dose pathogens 

such as Salmonella or growth of L. monocytogenes in perishable foods at refrigerated temperatures. Spore 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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forming bacteria survive cooking and pasteurization that are designed to kill vegetative pathogens; 

inadequate acidification, and/or temperature control have led to growth of toxigenic bacteria and been 

associated with foodborne illness. Parasites such as Cyclospora have also been associated with some raw 

agricultural commodities. However, there are no reliable testing methods for Cyclospora. 

2. How likely are ingredients to be contaminated, given the nature of the ingredient and the 

robustness of the supplier programs? The likelihood that ingredients are contaminated depends on the 

source of the ingredient and the potential exposure to contaminated environments (e.g., raw milk, grains, 

spices, plant-based materials grown in or harvested from the ground) and whether they have received a 

validated robust lethality process. Food ingredients that have been harvested or processed to minimize 

contamination (e.g., ingredient grown using good agricultural practices; use of sanitizers to reduce cross 

contamination between produce items) or receive some lethality step (e.g., irradiated spices, roasted 

peanuts) have a lower probability of being contaminated but often rely on supplier control programs to 

prevent post-lethality contamination. 

3. Are there robust processing control procedures such as a kill step or other reduction methods 

controls? Validated lethality steps such as thermal or high-pressure treatments (milk, juices), roasting 

(nuts/seeds), and baking (bakery) reduce the need for final product testing as a verification of preventive 

controls. However, even though vegetative microorganisms may be destroyed, control processes need to 

be in place to prevent growth of toxigenic organisms during production (e.g., B. cereus in batters, fillings) 

to ensure heat-stable enterotoxins are not present after cooking; hence in-process testing may be 

relevant in these circumstances. 

Even if a kill step is used sometime during processing, products that introduce ingredients postlethality (e.g., lettuce to a sandwich, herbs to cheese curd, icings on baked goods), particularly addition of 

ingredients that are raw or minimally processed, will be at higher risk for containing pathogens and may 

need testing of the individual component or the finish product. Products with a short shelf-life present 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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challenges for testing. While raw produce is washed, those washes do not necessarily achieve substantial 

microbial reduction in the food. Suppliers of produce to be consumed without a kill step need to comply 

with appropriate control measures to prevent or minimize pathogen contamination (for examples of 

control measures, see the Produce Safety Rule 21 CFR Part 112 (47)). 

Although thermal treatments are common microbial reduction steps, the formulation of a 

commodity may also reduce risk of microbiological contamination and hence the need for product testing. 

For example, cold-filled acidified foods, such as prepared mustards, hot-sauces, acidified cucumbers, or 

salad dressings made with vinegar, frequently rely on an acid-hold procedure for lethality as an alternative 

to thermal processing (6, 7, 25, 33, 42). In other foods, the acidity alone may not be sufficient to generate 

an appropriate (e.g., 5-log) kill of vegetative pathogens within several hours or days, but there may be a 

more gradual inactivation over time. Cultured dairy products, such as yogurt and sour cream, frequently 

have sufficient lactic acid production (e.g., pH decreases to <4.8 within 4-18 h) to inhibit growth of 

pathogens during production but also to generate additional inactivation (e.g., 1-log) during refrigerated 

storage (18, 19, 34). However, acid type also has an effect on lethality rate during thermal processing and 

for acid-hold lethality. For example, for foods acidified with citric acid, the killing may be relatively slow, 

whereas foods with predominantly acetic acid (such as pourable salad dressings) may result in shorter 

death times (1, 9, 42). Hard cheeses made with unpasteurized milk rely on a combination of high-quality 

milk, acidity (typically lactic or propionic acid), reduced moisture (aw), and extended aging for pathogen 

reduction, although there is evidence that more than 60-day aging may be required for safety (15, 16, 49). 

Other commodities with low aw (dried nuts/seeds) may also undergo slow pathogen reduction 

(17, 39). However, because the pathogen survival time may be measured in months, there likely is not 

enough time for sufficient reduction in pathogen numbers to exclude the need for product testing. 

4. Is there potential for microbial recontamination of product prior to packaging? Could there 

be pathogens due to environmental or handling contamination? Except for foods that are hot-filled, 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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filled within a closed system, or which receive an in-package lethality step, all commodities have the risk 

of contamination from handling or from the environment. 

5. Does the product formulation allow microbial growth or survival or cause death under 

conditions of transportation and various types of storage (refrigerated, frozen, ambient)? Microbial 

survival, growth, or death may occur as a result of intrinsic properties of the food, such as pH, acid type, 

water activity, salt levels, or formulation with preservatives or due to extrinsic properties such as 

packaging environment and transportation/storage temperatures. Verification testing may be indicated 

where storage conditions alone (freezing or refrigeration), rather than intrinsic properties of the foods, 

are the primary barrier to microbial growth, and process and environmental controls cannot ensure 

absence of the pathogen. For products that do not support growth of pathogens at ambient temperatures 

but have a history of post-lethality contamination by low-infectious dose pathogen (e.g., peanut butter, 

dry milk, chocolate), testing may be relevant to detect catastrophic failures (see appendices for examples). 

6. Is this product meant for higher risk (sensitive) population? In most of the example foods 

(Appendices A-F), the product is being made for the general population, but may be consumed by 

individuals in higher risk populations. Special considerations should be given to foods that are specifically 

manufactured for infants, elderly, pregnant, and immunocompromised or hospitalized consumers (e.g., 

milk powders used for infant formula and infant cereal, foods destined for nursing homes or hospitals). 

7. What is the shelf life of the product? Shelf life plays a role in the potential for microbial growth 

as well as timeframe in which testing results will need to be available before the product is distributed 

and consumed. The shelf lives of the example food products in this document range from several days to 

1-2 years. A longer shelf life increases the time available for microbial growth, potential for temperature 

abuse, and the risk that a consumer may eat a contaminated food (e.g., L. monocytogenes on soft 

cheeses). While short shelf life reduces the time for microbial growth under normal storage conditions, it 
 

 

 

 

 

 

 

 

 

 

 

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may be impractical to get results from pathogen testing of the food prior spoilage (e.g., being able to 

detect Salmonella in cut melon or STEC on leafy greens). 

8. Will consumer handling and use increase or decrease risk of pathogen survival, growth, or 

toxin production? Considerations should be given to the potential for abuse of the food by the consumer 

once it leaves the control of the manufacturer and retail chain. Does the consumer heat the food to 

reconstitute it or for palatability or eat it without further preparation? Is it likely that the consumer will 

hold a frozen food under refrigeration or hold a refrigerated food at temperatures greater than 4°C? How 

likely is a consumer to use a refrigerated food beyond the use-by date, particularly if the food is not grossly 

spoiled? 

Microbiological testing for verification of process control (as part of the facility’s food safety 

system) is different from microbiological testing for lot acceptance. 

Prior to widespread use of preventive controls, traditional microbiological testing has been lot 

testing for acceptance or rejection of that lot (i.e., to demonstrate that the lot is appropriate for its 

intended use). The purpose of lot testing is to examine a product lot for which you have no information 

(8). This testing can be useful when, for example, a government agency tests imports at the port of entry, 

or a food business tests an ingredient from a new supplier. Such testing should involve analysis of a large 

of number of samples randomly taken from the entire volume of food under consideration (8). Industry 

also uses “hold and release” testing for certain ingredients prior to use or in response to microbiological 

contamination issues. Such testing is useful to detect high rates of contamination, but it is not very 

effective when food safety systems are under control or to detect low rates of contamination. 

The purpose of microbiological testing for verification of process control is not to demonstrate 

that a lot of food is safe, but instead to demonstrate that control measures are functioning as intended 

(8). Rather than testing a large number of random finished product samples from a lot, a few finished 

product samples are taken from many lots on a regular basis (routine testing). Also, samples may be taken 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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at several intervals during production of a lot in order to detect contamination that may occur sporadically 

during production; often these are composited into one or more test samples. The results of the tests are 

analyzed to look for trends and to determine whether they meet an established criterion or indicate an 

out-of-control process. Testing may be conducted at a relatively high frequency initially to determine 

process capability. Past performance could be used to reduce the amount of testing over time (55). 

Microbiological testing of finished product for verification of process control can provide risk 

reduction, since the removal of any lots testing positive for a pathogen prevents that product from 

reaching the consumer. In addition, if investigations into the root cause of circumstances that led to the 

presence of a pathogen or to exceeding a process control criterion identify the source of the problem, this 

can be corrected, which will lead to the production of safer food in the future. 

Microbiological testing of finished product is most useful (1) if ingredients in a food have the 

potential to contain pathogens and there is no kill step (or a marginal kill step) in the manufacture of 

the finished product, and/or (2) when finished products is reasonably likely to be contaminated from 

the environment. 

Use of microbiological testing as a verification of control measures should consider risk to the 

consumer. Testing is more valuable if the pathogen of concern is likely to cause serious adverse health 

consequences or death, e.g., Salmonella vs. Staphylococcus aureus. Where there is a low risk to 

consumers, microbiological testing would be infrequent or there would be no testing. 

Microbiological testing should be increased when information indicates that the operation is 

not under control (e.g., records indicate a deviation at a critical control point, CCP, a pathogen has been 

detected on a food contact surface or in the finished product, a food has been involved in illnesses). 

A facility should consider the nature and extent of supplier control programs for ingredients 

and environmental monitoring programs in the facility in determining the role of finished product 

testing to verify control measures in a facility. In determining testing of finished product, a firm should 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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consider all programs in place to minimize the potential for the finished product to be contaminated. 

Having confidence that a supplier has implemented a robust program to minimize the potential for 

pathogens to be present in ingredients is one of the components of the food safety system being verified. 

Similarly, when the source of a pathogen in a finished product could be from the processing environment, 

having a robust sanitation and environmental monitoring program can significantly reduce the need for 

finished product verification testing. 

Sampling small amounts of product more frequently provides better information about process 

control than taking a larger sample equivalent in weight to the sum of the smaller samples. For example, 

taking small samples (e.g., 10-25g) on a frequent basis (e.g., every half hour) throughout a process run 

and testing a composite (e.g., 375 g, or multiple composites) provides more information on process 

control than taking a sample of the same weight (e.g., 375 g) from one or more packages, because 

contamination is generally expected to be nonhomogeneous and it provides a better picture across the 

day’s production (31). For certain commodities, such as dry dairy products, use of autosamplers are used 

to take samples throughout production and composite samples analyzed for target microorganism (43). 

Microbial test methods must be appropriate for the intended use (e.g., for detection of the test 

microorganism(s) in the specific food). To ensure reliable results, test methods should be validated to 

show they can detect the microorganism of concern in the specific food. For example, many spices have 

inhibitory properties, and the method used when testing the spice must consider this fact, e.g., by dilution 

of the inhibitors to the extent that the organisms of concern can grow. 

Microbiological testing for process control can be used to drive excellence in quality and process 

improvement. Testing for microorganisms that are in sufficient numbers to enumerate and then striving 

to reduce those numbers as low as possible can enhance product quality. Knowing the expected range of 

counts can identify when a change has occurred in the system (e.g., faulty practices) by detecting numbers 

that are outside the range; investigation as to why the numbers increased can lead to the identification 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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of a processing failure, an increase in microbial load in an ingredient, or another aspect of the process 

that warrants greater control. 

Charge Question 2. Are there situations in which testing other than for pathogens or indicator 

organisms, e.g., enzymes, would be an appropriate verification activity. 

Naturally occurring enzymes in raw commodities are heat sensitive and are therefore suggested as an 

alternative to use of other temperature-time monitoring to verify that a lethality step has been applied. 

However, the use of enzyme-based tests to verify the adequacy of processing is limited, particularly for 

multi-component foods. For enzymes to have practical application to be used as verification in lieu of 

product testing, they should: 

• Have inactivation kinetics in the processing range that are similar to those of the pathogens of 

concern. 

• Be consistently present at high enough levels such that the absence of detectable enzymatic 

activity does not occur before adequate inactivation of the pathogens of concern. 

• Not be reactivated within the timeframe needed for testing the food. 

• Be detected using procedures that are rapid, inexpensive, and easy to perform in a food 

processing setting. 

The inactivation kinetics of the enzyme determined in a food ingredient in which the enzyme is 

present may be different when the ingredient is combined with other ingredients, and thus may no longer 

reflect the inactivation of the pathogen of concern. Therefore, testing for indicator microorganisms may 

be more practical for process verification than testing for enzymes. 

Several non-microbial indicators have been identified. Alkaline phosphatase is used as an indicator of milk 

pasteurization (38, 45). Electron paramagnetic spectroscopy can be used to detect changes in cellulose in 

spices in response to gamma irradiation (40). Peroxidase has been used for validation of blanching in 

vegetable products (28). The peroxidases in carrots and potatoes maintained approximately 50% of their 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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activity after heating for a minute at 85°C (4); this time and temperature combination is considered to be 

generally sufficient to generate a 6-log reduction of L. monocytogenes in many food matrices (37). 

Thermostable deoxyribonuclease (TNase) is a product of pervasive staphylococcal growth; its presence 

indicates possible enterotoxin contamination in cheeses and sausages (24, 44). Other non-microbial 

testing verification activities may include monitoring of the rate of acid production (pH, titratable acidity) 

during production of cheese and cultured dairy products that assures adequate competition with 

pathogens to prevent growth during fermentation. 

Charge Question 3. Are there situations where verification testing would not be necessary if 

there is evidence that the appropriate treatment was, in fact, applied. 

For some foods, there is little or no benefit from microbial testing if validation and monitoring affirm that 

the lethality process is sufficiently robust and appropriately implemented, provided there is no 

opportunity for recontamination; in these instances, measuring processing parameters (e.g., temperature 

and time) provides adequate verification that pathogens have been controlled (e.g., foods in which a 

lethal treatment is delivered to product in the package). 

These foods include products that are processed (e.g., validated lethality process) and hot-filled 

or packaged under aseptic conditions in which contamination of the food after processing is prevented, 

or processed in the package (e.g., cook-in-bag). The use of “clean fill” technology for certain extended 

shelf-life foods, such as some beverages, yogurts, and desserts, can provide protection from 

recontamination. For aseptic and clean-fill foods, monitoring of the parameters of the process and 

verification activities other than finished product microbiological testing should be sufficient. 

There are also products in which the formulation is validated to be lethal to the pathogens of concern 

(e.g., vinegar, highly acidic juices such as lemon and lime, many mayonnaise or pourable acidified dressing 

formulations). Verification of formulation control (e.g., measurement of pH and total acidity) can provide 

appropriate evidence that those pathogens have been controlled. 
 

 

 

 

 

 

 

 

 

 

 

 

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For raw foods that are not subjected to a lethality step, and for foods that are subjected to postlethality handling with potential for recontamination, verification testing is appropriate. Some of these 

products include untreated spices, fresh fruit and vegetables, nuts, sandwiches, and deli salads. 

However, for most of the foods under consideration, food safety control will involve monitoring process 

parameters, ingredient testing, supplier audits, enforcement of employee hygienic practices, and a robust 

sanitation program verified in part by environmental monitoring/testing for microbiological indicator 

organisms, and records review that is supplemented by verification testing of food for pathogens or, more 

commonly, by indicator organisms. The extent of verification testing will depend on the confidence in the 

process, including how much safety is built into the process, and the other programs in place. 

Charge Question 4. When microbial testing is an appropriate verification activity, what 

considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

indicator organism) and type of test (e.g., presence/absence or enumeration)? What are appropriate 

indicator microorganisms for verifying processes that adequately control pathogens? 

A company considering conducting microbiological testing as a verification activity should include 

several factors related to the possible presence of microorganisms and the type of test. One fundamental 

question to address is whether to test for a specific pathogen or to test for another microorganism that 

can indicate the potential presence of the pathogen of concern or conditions that could lead to its 

presence. While microbiological testing for indicator organisms (e.g., aerobic plate count, 

Enterobacteriaceae, coliforms, or molds in product, or Listeria spp. or Enterobacteriaceae in the 

environment) does not necessarily mean that pathogens are present, trends of “out of spec” populations 

of these organisms indicate that investigations are warranted to determine root cause and to evaluate 

the impact on the safety of the food. 

In situations where microbial testing is deemed an appropriate verification activity, several criteria 

should be considered in selecting the microorganisms: 
 

 

 

 

 

 

 

 

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a. Which pathogens have been associated with the specific food or ingredient based on 

epidemiological and historical evidence? 

b. Is there a relevant indicator organism that is more likely to be present in a given commodity or 

processing environment than a pathogen (such as testing for Listeria spp. as an indicator for 

Listeria monocytogenes)? 

c. What impact do process steps have on the viability of pathogens or indicator microorganisms (is 

thermal process sufficient to kill STEC but allow lactic acid spoilage bacteria to survive; do spores 

survive the process; is there a potential for growth of microbes during extended runs)? 

d. What is the potential for recontamination of the food product after treatment and what are the 

microorganisms involved? 

e. What are the intrinsic and extrinsic characteristics of the food that may be conducive/selective 

for specific microorganisms to grow or survive? 

f. Is the food specifically intended for those individuals with higher susceptibility for infection to the 

pathogens of concern (e.g., hospital meals, infant foods)? 

g. What is the expected shelf-life of the food product? Is it practical to get microbiological tests 

before the end of shelf life and still market the product (e.g., hold-test for short shelf-life 

products)? 

The type of test to be used will depend on the validated microbiological methods available for a given 

matrix, as well as regulatory requirements. Enumeration of a pathogen in a food is appropriate when the 

risk of illness is related to the number of organisms present (e.g., B. cereus, C. perfringens, S. aureus). For 

low-infectious dose pathogens (e.g., Salmonella, some strains of Shiga-toxin producing E. coli, Cyclospora), 

some performance standards require detecting a single colony forming unit (CFU) in 25 g or more. Because 

routine plating methods are typically limited to detecting a lower limit of 10 CFU per g, many pathogen 

testing protocols are restricted to determining the presence or absence of the pathogen within a given 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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sample size. In the case of some pathogens, such as Cyclospora, enumeration methods do not currently 

exist. Although higher numbers of pathogens, such as Salmonella, reflect greater risk for consumers, 

enumeration is not needed to take action in response to positive findings. 

When food safety systems are under control, the presence of the pathogens of concern is not likely, 

and when present, they are likely to be heterogeneously distributed, and may be at a low level that is 

difficult to detect (31). Thus, testing for other non-pathogenic indicator microorganisms that are likely to 

be present more frequently and in greater numbers provides the advantage of being able to detect 

processes in which controls have not been adequately implemented or processes that are drifting out of 

control and thus are at increased risk of pathogens being present (8). The choice of indicator organism 

should consider if there is sufficient scientific evidence that the microbe is relevant for the food type and 

pathogen of concern (10, 14, 29, 30, 32). 

Trend analysis of indicator organism populations should be able to detect when controls may require 

corrections before pathogens become a problem or may indicate how likely that pathogen contamination 

has occurred; presence or populations of indicator organisms that exceed the preset limits requires 

investigation to prevent contaminated product from entering commerce (54). Depending on the results 

of testing the food (or environment) for indicator organisms, testing the food for the pathogen may be 

appropriate. 

Lastly, the type of testing selected should consider if there is a validated test for the pathogen of 

concern in the specific food matrix and the speed of detection that allows timely decisions regarding 

corrective actions or product disposition. 

Charge Question 5. What principles and criteria should a company apply in determining the 

frequency of testing finished product to determine if the company’s food safety system for that product 

is effective? 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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The frequency of testing for a finished product depends on a variety of factors, including ingredients 

used in the food, whether or not the food has had a validated robust lethality process, whether the food 

is packaged to prevent recontamination, whether the food is intended for a high-risk population, 

sanitation controls, and whether environmental monitoring suggests the potential of recontamination 

(see Appendices A-F of this document for specific examples). Buchanan and Schaffner (8) indicate that 

two key factors related to frequency of testing are the frequency at which a testing criterion will be 

exceeded and the response time that is needed in declaring a system is out of control, which are typically 

determined as part of a “process control study.” Testing more frequently will be more effective in 

identifying a loss of process control. Testing frequency should be increased when there is indication of 

loss of control in order to assist in root cause analysis and to more quickly determine when control has 

been restored (8). 

In the case of products with a terminal, validated lethality process in the package (e.g., cook-in-bag, 

high-pressure pasteurization of the package, or hot-fill) or those filled in a closed system (e.g., pasteurized 

milk), routine testing of finished product for pathogens may not be needed. Rather pathogen testing may 

be limited to situations where process control parameters are not met (e.g., when evaluating deviations 

for controls such as kill temperatures/time, cooling rate, or storage temperature). Typically, testing can 

be limited to spoilage microorganisms that are indicators of shelf-life related to quality of ingredients used 

or additional verification of process control such as such as Pseudomonas spp. in pasteurized milk or lactic 

acid bacteria in cook-in-bag products. 

For products that have a microbial reduction processing step but that are subsequently exposed to 

the environment prior to packaging (e.g., products made with roasted nuts, butter or soft cheeses made 

with pasteurized cream or milk, baked cakes), lot testing for indicator organisms is frequently used as the 

primary verification of process control (see appendices for examples). Pathogen testing of finished 

product may be useful as a periodic check for process control (such as quarterly or as risk assessed). More 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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frequently, finished product pathogen testing is indicated if investigative testing from an Environmental 

Monitoring Program (EMP) for Listeria or Salmonella, suggests there is potential cross-contamination to 

the product from the environment, either inherently due to design and construction of the facility or 

equipment or due to the recurring presence of these pathogens in zones 2 or 1. In these cases, the 

implicated product is held and tested for the pathogen using a statistically based sampling program and 

validated detection method to determine contamination. 

However, in cases of short shelf-life foods (e.g., prepared sandwiches, cut melon, deli salads), testing 

of finished product for pathogens is impractical because the held product may be at the end of shelf life 

by the time results are confirmed. For these types of products, supplier control programs and EMP are 

more effective than finished product testing for pathogens. Microbial testing of product is focused on 

trending indicator organisms to identify loss of process control as a supplement to supply chain control 

for ingredients and robust sanitation/environmental controls (refer to appendices for examples). 

For most products considered in this document, that have a long shelf stable shelf-life, unless there is 

a loss of process controls during production, environmental monitoring indicating a problem, or 

breakdown in supplier control programs, finished product testing might consist primarily of periodic 

testing for spoilage organisms for shelf-life verification or for microbial indicators of loss of process control 

(including sanitation processes). 

One situation where pathogen testing of RTE foods or ingredients with a long shelf life may be 

appropriate is for products that have a history of microbial contamination (e.g., milk powders). In these 

cases, hold and testing may be frequent, such as for lot-disposition. In general, the frequency of lot testing 

of the final product is determined by an assessment of risk. If the time for processing after lethality is long 

(such as days), or if product has multiple points of exposure to recontamination after the lethality step, 

frequency of testing will be greater than if the product is rarely handled and risk of exposure is limited. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Charge Question 6. Generally microbial testing by a company to verify process control is conducted 

on “finished product.” Are there situations in which testing at sites other than at the end of the process 

can achieve the goal of verifying the adequacy of control of microbial hazards? Describe the situations 

and the testing that would be appropriate. 

There are situations where testing or verification other than microbial testing at the end of the process 

(i.e., finished product testing) can achieve the goal of verifying the adequacy of microbial hazard control 

(see Table 2 for comparison of testing for various commodities and Appendices A-F of this document for 

details). Alternative sites and strategies include, but are not limited to, ingredient testing by suppliers or 

processors, robust environmental monitoring, and in-process product measurement of food qualities 

(such as rate of acid development during fermentation) that affect microbial growth. Selection of 

strategies will be influenced significantly by commodity/food characteristics (for example pH or aw values 

in food that are able to support growth vs. being inhibitory), use of a validated microbial kill-step, and the 

degree of post-lethality handling. 

In some cases, an ingredient is used in manufacturing a food where there is no additional control 

applied for a hazard associated with that ingredient. In such instances, microbiological testing of the 

ingredient prior to use can be an important measure in ensuring control of a hazard. Such testing is often 

conducted by the supplier (usually the supplier contracts with an independent accredited laboratory for 

the testing) and a certificate of analysis (COA) is provided to the customer. COAs provide assurance of the 

suppliers’ control processes at the time of sampling and testing. COAs may not be needed for each 

shipment of an ingredient. The frequency of such testing depends on many factors, including the 

likelihood and severity of illness if the hazard were present in the ingredient, knowledge about the food 

safety system implemented by the supplier (e.g., obtained through an audit), and the safety history of the 

ingredient received from the supplier. It is recommended that testing ingredients from a supplier be 

periodically performed by the customer to verify the efficacy of the supplier’s control programs. The 
 

 

 

 

 

 

 

 

 

 

 

 

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frequency of periodic testing should provide confidence that suppliers’ programs are indeed effective. 

Written procedures for the sampling plan should include how to collect and prepare the samples, and 

describe the analytical methods used. Testing of ingredients is not warranted when the manufacturer uses 

the ingredient in a product for which there is a process control measure that would address that hazard 

(e.g., a kill step), unless the manufacturer’s control measure is dependent on the ingredient containing a 

low pathogen load (which could be reflected by samples testing negative for a pathogen). 

Testing of food characteristics such as pH or aw can also be performed on in-process product or 

finished product and can replace microbiological testing of finished product. For example, during a 

fermentation process, the pH of in-process product could be measured to monitor the acid production 

that can control microbial hazards. When characteristics such as pH and aw are relevant to the safety of 

the product, periodic testing intervals of the food product batches should be established. Using food 

characteristics as process control parameters requires establishing and maintaining records to include 

equipment calibration, monitoring and verification of the parameters, review of the process control 

records, and any corrective actions. As noted above, the rapid reduction of pH may be important in 

controlling pathogen growth in a food fermentation process; similarly, the reduction of moisture or aw 

during a drying process may be important to monitor. If these steps are under control, testing for 

pathogens such as S. aureus or B. cereus or their enterotoxins (if these are a concern for the products) 

would not be needed. 

Testing of product during validation studies of process controls can provide the data needed to show 

that microbiological hazards of concern can be consistently controlled. The microbiological data obtained 

during validation prior to implementing a process and during the initial stages of implementation to 

demonstrate consistent control may indicate that finished product testing is not needed as long as the 

monitoring of the process parameters that were validated indicates the process remains in control. 
 

 

 

 

 

 

 

 

 

 

 

 

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Charge Question 7. The CGMP & PC rule requires environmental monitoring for an environmental 

pathogen (e.g., Listeria monocytogenes, Salmonella) or for an appropriate indicator organism as a 

verification activity if contamination of an RTE food with an environmental pathogen is a hazard 

requiring a preventive control (such as sanitation controls). What impact does environmental 

monitoring have on frequency and extent of product testing verification activities by companies? 

Environmental monitoring as a verification of sanitation controls is more effective than solely testing 

finished product, but it may not eliminate the need for finished product testing. The results of 

environmental monitoring could indicate that product contamination may have occurred (e.g., a product 

contact surface tests positive for Listeria spp. and follow-up tests indicate the potential for product 

contamination) and this could lead to product testing as part of actions to identify the root cause and 

correct the problem (52). 

Determinations of potential harborage sites for pathogens through periodic testing for the pathogen 

or an indicator organism (e.g. food contact surfaces, zone two is non-food contact surfaces in close 

proximity to food contact surfaces, zone three is non-food contact surfaces not proximal to zone one, and 

zone four is areas remote from production) is recommended (12, 13, 20, 26, 27, 41, 52). Samples should 

be taken several hours into processing, or at the end of the day prior to sanitation. The degree of 

environmental monitoring is impacted by, but not limited to product characteristics, process type (wet v. 

dry), facility and equipment design, process and product history, supplier monitoring program, and target 

of environmental program (indicator, pathogen, non-microbial). Manufacturers should refer to 

commodity-specific guidance for environmental monitoring programs (2, 11, 21, 22, 26, 27, 52). While 

Salmonella is frequently the target pathogen for control in dry environments and Listeria monocytogenes 

in wet environments, both microorganisms may need to be considered in many processing environments. 

Environmental monitoring can influence frequency and extent of product testing. An Environmental 

Monitoring Program (EMP) should be designed to detect pathogens or indicator organisms in zones one 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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and two or other areas that pose a risk of cross-contamination to product. When contamination of an RTE 

food by Salmonella or Listeria monocytogenes from the processing environment is a primary concern, a 

robust EMP should reduce the need for product testing (e.g., frequency, number of samples). This is 

particularly the case for RTE foods that receive a validated lethality treatment but may subsequently be 

exposed to the environment (e.g., after the lethality treatment but prior to final packaging) where crosscontamination is possible. Examples of RTE foods where EMP can reduce the need for final product testing 

include cheeses made from pasteurized milk, butter, cultured dairy products, dried dairy products, ice 

cream, roasted nuts and nut products (for summary, see Table 3; details are found in Appendices A-F of 

this document). 

For some food products, an EMP is the primary means for verification of effective sanitation control 

programs and finished product testing is not typically conducted unless triggered by other data (e.g., zone 

1 or zone 2 environmental positives). Examples here include RTE baked items (time-temperature 

controlled for safety, TCS, and non-TCS), RTE cereals, RTE grained-based baked products, RTE cold pressed 

bars (Appendix B), RTE meals and sandwiches with short shelf life (Appendix C), and fresh cut fruits and 

vegetables with short shelf life (Appendix E). 

In some cases, an EMP is implemented in conjunction with routine finished product testing, although 

the results from the EMP may still influence the degree and level of finished product testing. For example, 

there are regulatory requirements for finished product testing for powdered infant formula (i.e., 

powdered infant formula must be tested for Cronobacter spp. (30 X 10 g) and Salmonella spp. (60 X 25 g) 

in accordance with 21 CFR 106.55). Powdered infant formula may be subject to contamination by 

Cronobacter spp. from the environment and an EMP may indicate the need for additional product testing 

for Cronobacter. Other examples of products where both an EMP and routine finished product testing is 

appropriate could include raw milk cheeses, certain soft cheeses (e.g., soft ripened; Appendix A), RTE nuts 

not processed for lethality (Appendix D), and nut butters (Appendix D). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Charge Question 8. (1) What criteria should a company apply in determining that microbial testing 

results indicate a loss of process control? (2) What actions should a company take if test results indicate 

a loss of process control? (3) When verification testing indicates loss of process control, to what extent 

should verification testing be increased, how far upstream and downstream should it go, and when and 

how should it be scaled back? 

Answer Q8 -1. What criteria should a company apply in determining that microbial testing 

results indicate a loss of process control? 

For this document, process control refers to the entire operation (e.g., entire food safety 

system/process). It is not restricted to process preventive controls. 

A food safety system and the manufacturing process managed by that system are in control when, 

within the limits of a stable and predictable process variation, all food safety hazards are controlled to an 

acceptable level. Building on this definition, the development of measurable attributes that indicate 

whether a process maintains or surpasses an acceptable degree of hazard control or falls below that level 

is required (29). 

One measure of process control is the adherence to microbiological limits established in the food 

safety system for verification of activities such as those used for sanitation and processing controls 

intended to mitigate microbiological hazards. Failure to meet prescribed microbiological testing limits for 

indicator organisms or pathogens could constitute a loss of control. A food manufacturer should 

determine limits relevant to its specific products and processes. Guidance, not regulatory limits, is 

provided in this section and in Appendices A-F. 

The measurable attribute and the type of microbial testing used to measure process control will 

vary with the product, the hazard being controlled, and the location of the control along the process 

continuum. Once actionable limits for test results are established at points along the entire manufacturing 

process, a company can then respond to those results based on food safety impact. 
 

 

 

 

 

 

 

 

 

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Measurement of process control is based on the following (35). 

1. Sampling and assessing the output of the process for key microbial targets should occur at a 

frequency that limits the amount of time that a loss of control goes unrecognized. Frequency of 

sampling is predicated on the propensity for the system to lose control, the prevalence of the 

microbial target and practicality, balancing rapid recognition of a system out of control with the 

cost of sampling and testing. Sampling sites are selected that are representative of the product as 

it passes through the process or as it exits the process. Larger sample sizes add statistical 

relevancy. Testing frequency and sample size taken should be risked based. More intensive 

testing is needed for foods which there is little information, e.g., for new suppliers, a new 

processing line or product, or for individual foods or ingredients that have been shown to have 

higher prevalence of microbial risks e.g., for spices obtained in certain regions. As a firm builds 

data base of microbial results, can refine understanding product will be outside microbial limits 

that have been identified to verify that process is in control. 

2. Process control performance limits and testing targets (e.g., specifications) are predefined for the 

type of food product, type and extent of processing, RTE status, chemical and physical 

characteristics of the food product, and the history of the process. Microbial criteria for food 

safety or food quality need to be relevant to signaling a hazard in a specific product and be 

attainable. 

3. A system for documentation and review of results is in place that allows corrective action with 

the appropriate level of immediacy. 

4. A predetermined plan of action (POA; a corrective action plan) is developed based on a scaled 

response considering public health impact, deviation from relevant limits, and frequency of the 

deviation. For example, a typical set of POA choices might be take no action, move to increased 

sampling frequency or sample size, conduct a predetermined internal or external audit of the 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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process that is typical for out-of-control variability, and identify an assignable cause through rootcause analysis and take corrective and preventive actions. The corrective actions specified must 

be subsequently verified to ensure they reduce or prevent future deviations. The proper action 

should be decided upon based on the severity and frequency of the deviation. 

5. The microbial measurement of insanitary conditions through environmental testing could also 

indicate the loss of process control or contribute to an overall assessment of loss of control. 

An adequate process control indicator is an attribute that can be measured with objectivity and for 

which limits that indicate a need for corrective action can be established. The primary strength of process 

control indicators is signaling the need for a more comprehensive analysis of the system and to take 

corrective action before a noncompliance occurs. An ideal indicator of process control is one that allows 

corrective actions to be taken before a loss of control represents a threat to public health. USDA FSIS 

reviewed the use of process indicators in its public health risk-based inspection system (29). The agency 

proposed two basic types of process indicators: those that may predict a future loss of control (e.g., 

exceeding a specific rate of out of specification (OOS) results) and those that reveal outcomes of a past 

loss of control (e.g., finding a pathogen in an RTE food product, recall of a product for safety reasons). 

Limits (criteria) that are chosen as indicators of process control should take this distinction into 

consideration, as the type of process control indicator will determine the criticality of the corrective 

action. For instance, the presence of an indicator organism could reflect normal variation within 

acceptable parameters and not necessarily demonstrate that a process is out of control. In this case, the 

frequency of finding an OOS result becomes important in determining loss of control. However, the finding 

of a pathogen-contaminated product indicates an overt loss of process control that could have occurred 

in the past, unrecognized by the facility or inadequately addressed by actions taken in response to a prior 

failure. 
 

 

 

 

 

 

 

 

 

 

 

 

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The following factors should be considered when analyzing an OOS result and determining whether a 

loss of process control has occurred. These include, as appropriate: 

• the target organism and levels detected, i.e., a qualitative pathogen (e.g., presence of Salmonella 

in a 375 g sample or environmental sample), quantitative pathogen (e.g., the number of 

Staphylococcus aureus) or an indicator organism (e.g., the number of coliforms). 

• the type of sample analyzed, i.e., ingredient, in-process, environmental or finished product. 

• location of the sampling site and proximity to finished product. 

• to what extent did the level deviate from the limit for a quantitative microbiological result? 

• frequency with which OOS results are obtained. 

All or some of these factors can be used to determine a level of criticality that will drive scalable 

reactions from recleaning a piece of equipment to discarding product. For instance, the finding of a 

pathogen in product or in close proximity to product would warrant an immediate and aggressive reaction 

as compared to an OOS indicator level in in-process product. 

Identifying and ranking process control indicators can be challenging. The relative importance of 

different predictors will vary with the products produced, the state of the processing facility, raw 

ingredient sources and several other variables. Appendices A through F in this document describe six 

commodity groups and provide a comparison of microbial limits for determining whether processes are 

out of control depending on the product manufactured. Two examples of microbial limits drawn from 

Appendices A and D are shown below. Additional information on establishing microbiological safety 

criteria can be found in Scientific Criteria to Ensure Safe Food (36). 

Example 1. Appendix A - Dairy Products. 

When there is a loss of systemic process control for soft cheeses as recognized by the finding of a 

pathogen in product or a frequent occurrence of OOS indicator organism results, a root cause analysis 

should be performed, including looking at heat-treatment of milk, cheese vat/make procedures, 
 

 

 

 

 

 

 

 

 

 

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705 acidification rate, finishing table, brine tanks, block formation, aging, cutting, and packaging to determine 

706 the source(s) of loss of control and to implement corrective action. The findings of the root cause analysis 

707 will dictate corrective actions and whether verification testing that includes finished product is indicated 

708 (Appendix Table A-1). 

709 

710 Example Appendix Table A-1. Microbial targets, limits, and recommended actions if limits are exceeded, 

711 for soft cheeses made with pasteurized milk. Additional testing may be indicated for cheeses made with 

712 raw milk (5, 23). 

Target 

Microorganism 

Microbial 

Limit 

Recommended Action if Limit is 

Exceeded 

Comments 

Coliforms or 

Enterobacteriaceae 

<100/g Investigate reason for exceeding 

limit and implement corrective 

action; consider testing for E. coli 

(>10/g) if coliforms are detected 

Routine testing 

S. aureus <100/g If >104/g, reject lot due to 

potential for enterotoxin 

production. Due to heat stability 

of enterotoxin, diverting to further 

processing is not recommended 

Investigative testing if 

routine pH monitoring of a 

vat during fermentation 

suggests acid 

development is slow and 

culture is not active. 

Investigate, implement 

corrective action 
 

 

 

 

 

 

 

 

 

 

 

 

 

NACMCF RTETesting_MainText_Final_11Jul2021.docx 32 of 44 

Target 

Microorganism 

Microbial 

Limit 

Recommended Action if Limit is 

Exceeded 

Comments 

Listeria Negative in Reject lot. Investigate cause of Investigative testing as 

monocytogenes 125 g 

analytical 

units (5 x 25-

g samples) 

contamination. Determine if other 

lots are involved. Determine steps 

to prevent reoccurrence. 

response to EMP that 

suggests likely 

contamination of product 

or routine testing for 

products that can support 

growth of L. 

monocytogenes 

Salmonella Negative in 

375 g 

analytical 

units (15 x 25 

g samples) 

Reject lot. Investigate cause of 

contamination. Determine if other 

lots are involved. Implement 

corrective action to prevent 

reoccurrence. 

Investigative testing as 

response to EMP that 

suggests likely 

contamination of product 

or routine testing for 

cheeses made with raw 

milk and aged for 60 days 

713 

714 Example 2. Appendix D - Nuts (including tree nuts and peanuts) and Nut/Seed Products. 

715 Microbiological limits for Ready-to-eat (RTE) chopped raw tree nuts. Producers of RTE chopped raw tree 

716 nuts and some types of whole RTE nuts rely on preventive controls that include sanitation controls and a 

717 supply-chain program. Control is based on the expectation that processers beyond the grower are 

718 compliant with sanitation and supply-chain programs under the Preventive Controls for Human Food Rule 

719 (21 CFR Part 117)(51) and that growers that supply the raw unprocessed nuts are compliant with the 
 

 

 

 

 

 

 

 

 

 

 

 

 

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720 Produce Safety Rule (21 CFR Part 112)(50), where applicable, and Good Agricultural Practices (GAPs) (53). 

721 Finished product testing is conducted to verify that sanitation controls are in place and effective within 

722 the manufacturing facility. Product testing for Salmonella and generic E. coli provides highly relevant 

723 verification data and is appropriate for the level of risk associated with the raw nuts. One indication of 

724 loss of control would be the finding of a positive pathogen result. When a pathogen is detected from a 

725 sample taken at the end of the production line, the recommended action is to divert for reprocessing with 

726 a kill step or destroy the lot of raw nuts represented by the sample, as appropriate. 

727 The repeated finding of an indicator organism such as generic E. coli above a threshold level can 

728 also indicate a loss of sanitation control and the potential for pathogen ingress into the process. However, 

729 in this case, testing provides an opportunity to adjust the process and avoid public health implications. 

730 Actions taken would follow a tiered approach based on numbers and frequency of occurrence (Appendix 

731 Table D-1). 

732 Example Appendix Table D-1. Microbial targets, limits, and recommended actions if limits are exceeded, 

733 for ready-to-eat nuts not processed for lethality. 

Target Microorganism Microbiological Limit Recommended Action 

if Limit is Exceeded 

Comments 

E. coli (generic) <0.36 MPN/g Investigate, implement 

corrective action 

If 2 of 10 samples are 

>0.36 MPN/g, follow 

CPG Sec 570.450 (48) 

Listeria monocytogenes Negative in 25 g 

Reject. Investigate and 

implement corrective 

action 
 

 

 

 

 

 

 

 

 

 

 

NACMCF RTETesting_MainText_Final_11Jul2021.docx 34 of 44 

Reject. Investigate and Two 375 g analytical 

Salmonella Negative in two 375 g 

implement corrective units derived from 30 x 

samples 

action 25 g samples 

734 

735 Answer Q8-2. What actions should a company take if test results indicate a loss of process 

736 control? Microbiological and chemical limits for foods for use by the United States Department of Defense 

737 to assess process control and insanitary conditions were evaluated and published by a previous NACMCF 

738 committee (35). The microbiological limits reported for indicator organisms in that document are not lot 

739 acceptance criteria, unless there is a regulatory limit associated with that value, such as limits for coliforms 

740 in milk or generic E. coli in nuts (see NACMCF-DOD Appendices (35). The 2018 NACMCF-DOD document 

741 was developed for inspectors or auditors to evaluate whether a food was produced under sanitary 

742 conditions without having full knowledge of the processing conditions. However, the target 

743 microorganisms and limits included both product and environmental monitoring that would be useful to 

744 the manufacturer that their process is in control. Therefore, both the NACMCF-DOD guidance and this 

745 document provide guidance to evaluate sanitary conditions and process control for foods, including 

746 appropriate target microorganisms and limits in foods, as well as recommended actions to be taken if the 

747 limits are exceeded. In many instances, actions include investigating to determine a root cause, 

748 implementing corrective and preventive actions, and conducting follow-up sampling and testing to 

749 determine if the corrective and preventive actions have been effective. These actions were categorized 

750 as “Investigate” or “Implement Corrective Actions.” The 2018 NACMCF-DOD document indicated that 

751 investigative and corrective action procedures would likely be unique to each situation. Given the scalable 

752 approach recommended for determining loss of control, actions taken would also depend on the type of 

753 hazard created by a loss of control. 
 

 

 

 

 

 

 

 

 

 

 

 

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As an example, samples taken of a low water activity product (e.g., a cold pressed bar) at several 

in-process points during production are found to be out of specification for coliforms; however, levels 

decrease over the course of the process run. If the process had been wet cleaned prior to start-up, the 

investigation might focus on water left behind due to inadequate drying and outgrowth on the equipment 

and/or a review of coliform levels in ingredients. The fact that the coliform levels decreased over time 

would appear to support elevated levels due to outgrowth at start-up that were removed as the process 

progressed. The company could take the following actions: 

1. Review sanitation activities and implement corrective actions if found inappropriate or 

inadequate (e.g., modify cleaning and sanitizing procedures, revise sanitation verification 

activities). 

2. Review coliform levels in ingredients and implement corrective actions if found to be elevated 

beyond the ingredient specification (e.g., address issue with supplier, use alternative supplier). 

3. Consider whether pathogen testing of finished product could be appropriate. (As an indicator 

of post-process contamination, high levels of coliforms might also indicate a pathway for 

pathogen ingress). 

4. Decide on product disposition. 

In another example, samples are taken at the end of the production line and tested for a target 

pathogen. If the pathogen is detected, this represents a serious loss of process control that warrants 

stopping the process line until a root analysis is completed, the hazard is mitigated, and the hazard is 

assured to be eradicated. The root cause analysis could include a review of all processing records, 

questioning production workers about whether there were any unusual occurrences during processing, 

testing ingredients for the pathogen, environmental sampling, additional testing of product from 

throughout the production, etc. Specific corrective actions depend on the findings of the root cause 

analysis. Unless the product can be reprocessed using a validated process, product destruction is 
 

 

 

 

 

 

 

 

 

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indicated. An essential activity is to assess whether contaminated product has left the company’s control 

(public health risk) and take the necessary actions to recall the product. 

Answer Q8-3. When verification testing indicates loss of process control, to what extent should 

verification testing be increased, how far upstream and downstream should it go, and when and how 

should it be scaled back? The number of in-process, finished product, or environmental samples to take 

and test on a routine basis is determined by a review of the process and product, and the information 

derived from the analysis. In general, taking more samples increases the probability of pathogen 

detection; and larger numbers of samples taken for pathogens can increase the confidence of detecting 

pathogens present at a low prevalence. Analytical unit weights for testing should be a minimum of 25 

grams; for pathogen testing, the analytical unit is usually a composite weight such as 375 grams (15 X 25 

gram samples to result in a 375 gram analytical unit) (3) When there has been a loss of control, the number 

of samples, the size of the sample, and the frequency of verification testing can all increase. 

If a root cause is not readily apparent, investigational testing should span the entire process, 

including ingredient, in-process product and a sampling of finished product produced over contiguous 

runs or produced during a time frame bracketed by breaks in the process for full sanitation (“clean 

breaks”). The intent is to find ingress points and establish a timeframe for the contamination event. 

When a root cause investigation and corrective/preventive activities are completed, the decision to 

resume normal production is based, in large part, on microbiological testing that verifies control has been 

restored. Predetermined testing strategies (frequency and numbers of samples) for a process in control 

(standard “surveillance” level of testing), a process trending away from control (increased “heightened” 

level of testing) and a process that is out of control (investigative testing) should be part of a 

microbiological testing program. The increased number of samples and the frequency with which they are 

taken to initially investigate the root cause can be scaled back in a stepwise manner, first to a heightened 

level of microbiological testing and, eventually, to fewer samples, smaller sample sizes and fewer sample 
 

 

 

 

 

 

 

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sites consistent with surveillance testing used with a process in a steady state of control. This step-down 

approach requires a commitment to testing at each step for a defined amount of time to collect sufficient 

data that demonstrates the process is moving toward a consistent state of control. 

SUMMARY AND CONCLUSIONS 

This document provides examples and advice for manufacturers/processors to establish their own 

microbial targets and limits to meet the preventive control requirements about using microbiological 

testing for pathogens (or appropriate indicator organisms) to verify process control for pathogens in RTE 

foods under FDA’s jurisdiction. These decisions are made by each firm based on their facility, ingredients 

used, processing, packaging, level of anticipated control, shelf life of the product, intended use, or 

potential storage and handling at retail or by the consumer. 

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involving the presence of the organism Escherichia coli. https://www.fda.gov/regulatoryinformation/search-fda-guidance-documents/cpg-sec-570450-tree-nuts-adulteration-filth-involvingpresence-organism-escherichia-coli. 

49. U.S. Food and Drug Administration. 2016. FY 2014 – 2016 Microbiological sampling assignment 

summary report: raw milk cheese aged 60 days. https://www.fda.gov/media/99340/download, Accessed 

March 20, 2021. 

50. U.S. Food and Drug Administration. 2021. 21 CFR Part 112—Standards for the growing, harvesting, 

packing, and holding of produce for human consumption. https://ecfr.federalregister.gov/current/title21/chapter-I/subchapter-B/part-112?toc=1, April 11, 2021. 

51. U.S. Food and Drug Administration. 2021. 21 CFR Part 117—Current good manufacturing practice, 

hazard analysis, and risk-based preventive controls for human food. https://www.ecfr.gov/cgi-bin/textidx?tpl=/ecfrbrowse/Title21/21cfr117_main_02.tpl. 

52. U.S. Food and Drug Adminstration. 2017. Control of Listeria monocytogenes in Ready-To-Eat 

Foods: Guidance for Industry. https://www.fda.gov/files/food/published/Draft-Guidance-for-Industry--

Control-of-Listeria-monocytogenes-in-Ready-To-Eat-Foods-%28PDF%29.pdf, Accessed April 26, 2021. 
 

 

964

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966

967

968

969

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53. Western Agricultural Processors Association. 2015. Good Agricultural Practices (GAPs) For 

Production of Tree Nuts. https://ccgga.org/wp-content/uploads/2016/11/Good-Agricultural-Practicesfor-Nut-Growers_WAPA-9-11-15-Final.pdf, Accessed February 5, 2021. 

54. World Health Organization, F. 2019. Statistical aspects of microbiological criteria related to foods: 

A Risk Manager's Guide. https://apps.who.int/iris/rest/bitstreams/1060496/retrieve. 

55. Zwietering, M. H., L. Jacxsens, J. M. Membre, M. Nauta, and M. Peterz. 2016. Relevance of 

microbial finished product testing in food safety management. Food Control. 60:31-43. 
 

 

 

 

 

 

 

 

 

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1 APPENDIX A - CATEGORY: DAIRY 

2 Apart from some cheeses, the dairy food categories listed below are made with pasteurized milk 

3 to eliminate common vegetative bacterial pathogens. Therefore, the presence of any pathogen represents 

4 post-process contamination, loss of process control, or insanitary conditions. Salmonella, E. coli O157:H7, 

5 and L. monocytogenes are considered adulterants in RTE dairy products. In the United States, some dairy 

6 products such as fluid milk and yogurt are regulated by States under the Grade “A” Pasteurized Milk 

7 Ordinance (PMO)(63). Pasteurization of milk and milk products is required under 21 CFR 1240.61. FDA has 

8 enforcement policies for microbial pathogens and indicators of inadequate pasteurization or post9 pasteurization contamination of dairy products identified in the dairy compliance guidelines (64). 

10 Pathogens such as Salmonella and E. coli O157:H7 must be absent. Actionable limits for S. aureus and B. 

11 cereus are set at 104 CFU/g, whereas limits for generic E. coli or coliforms are product specific (64). Other 

12 resources for microbiological specifications and guidelines include the Compendium of Methods for the 

13 Microbiological Examination of Foods (4) and the Standard Methods for the Examination of Dairy Products 

14 (43, 72). Alkaline phosphatase concentrations in dairy products other than cheese and related cheese 

15 products are limited to less than 2.0 micrograms phenol equivalents per gram in one or more subsamples, 

16 whereas cheeses may have higher limits (1, 64). In many cases, coliforms or Enterobacteriaceae are 

17 acceptable for routine testing (40). The widely used microbiological count method is the standard plate 

18 count (SPC; usually referred to as aerobic plate count or APC) agar method that estimates the microbial 

19 population in various dairy products to determine contamination during processing. The SPC is the 

20 reference method standard by the National Conference on Interstate Milk Shipments (NCIMS) and PMO 

21 for raw and pasteurized milk and milk products (72). 

22 

23 
 

 

 

 

 

 

 

 

 

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40

45

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24 Dairy Products 

Butter, margarine 

26 Cheese, hard (e.g., Cheddars), extra hard, grating (e.g., Parmesan, Romano) 

27 Cheese, fresh (Queso fresco), soft, soft-ripened (Camembert), semi-soft (Edam, Gouda), veined 

28 cheeses (Roquefort, Gorgonzola) 

29 Cultured, pH < 4.8 

Cultured, pH > 4.8 and <5.4 

31 Dried products (including dairy ingredients used to make infant formula) 

32 Frozen desserts 

33 Milk and milk products (fluid) 

34 

Butter, Margarine 

36 Examples include sweet cream butter (salted and unsalted), cultured sour butter, whipped butter, 

37 whipped butter with herbs. For the purposes of this document, the Committee did not include examples 

38 for margarine. While margarine may mimic butter in appearance and use, the product composition, 

39 production methods, and microbial ecology are different than butter. Principal methods for controlling 

pathogens in butter are microbial quality of ingredients, pasteurization of raw materials (cream and milk), 

41 hygiene during production and packaging, minimal size and even distribution of the water droplets in the 

42 fat matrix, and the presence of salt (41). Starter cultures, if used, are not typically a source of 

43 contamination. Ingredients such as salt, coloring agents, and neutralizers are usually free of microbial 

44 contamination. Water used post pasteurization (e.g., for washing) should be of potable quality. 

Ingredients should be sourced from suppliers meeting specifications. 

46 
 

 

 

 

 

 

 

 

 

 

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47 Question 1. What principles and criteria should a company apply in determining the need for and in 

48 designing an effective microbial testing program to verify that processes are effectively controlling 

49 microbial pathogens? 

50 

Criterion/Factor Butter 

A. Are pathogens associated Yes. Outbreaks due to L. monocytogenes and S. aureus have occurred 

with the food or (24, 28, 44, 45, 49). Also, of concern are Salmonella, Campylobacter, 

ingredients? and E. coli O157:H7, but these pathogens are less likely unless adjunct 

ingredients such as herbs are added after the cream pasteurization 

step (19, 62) . 

B. Are the ingredients likely 

to be contaminated? 

No. Cream is pasteurized to inactivate vegetative pathogens. If fresh 

herbs are added to whipped butter, they could be contaminated with 

pathogens such as pathogenic E. coli, Salmonella, and Cyclospora. 

C. Are there robust 

processing control 

procedures such as a kill 

step or other reduction 

methods/controls? 

Yes. Critical ingredients such as cream and milk are pasteurized. 

Verification of supplier control of biological hazards should be used 

for any ingredient added post-lethality. 

D.Is there a potential for Yes. Butter is made with pasteurized cream, but churning, salting, 

recontamination from and packaging occurs after pasteurization. Product may be exposed 

the handling or the to pathogens from employees during handling and from the 

environment? environment. 
 

 

 

 

 

 

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Criterion/Factor Butter 

E. Does the product support Yes, studies have shown the potential for pathogens such as S. aureus 

survival or growth? or L. monocytogenes to grow in butter are impacted by the 

characteristics of the butter (pH, aw, salt, cultures), as well as the 

storage temperature (24, 28, 47, 71). Microbial spoilage of butter is 

caused mainly by yeasts and molds, and sometimes bacteria. These 

may be introduced through poor hygiene before or during packaging, 

or during use. Whipped butter made by the addition of milk, water, 

or incorporation of herbs can alter the emulsion and thereby support 

the growth of pathogens. Whipped butter made by the addition of 

gas only, does not significantly alter the ability of pathogens to 

survive or grow compared to unwhipped butter. Unsalted butter that 

has a higher water activity than salted butter can have increased 

microbial stability if pH is reduced as a preventive control during 

production. 

F. Is this product meant for 

higher risk population? 

Butter is not specifically intended for higher risk populations but will 

be consumed by the elderly and immunocompromised. 

G.What is the shelf life of 

the product? 

Refrigerated shelf life of butter varies between 3 and 9 months, 

depending on the level of salt, pH, or other preservatives present. 

H.Will consumer handling 

and use increase or 

decrease likelihood of 

The scenarios are unlikely to affect risk for salted butter but holding 

some types of unsalted or whipped butter at non-refrigerated 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Butter 

pathogen survival, 

growth, or toxin 

production and risk of 

consumer illness? 

conditions could allow growth of pathogens such as S. aureus if it is 

present. 

51 

52 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

53 enzymes, would be an appropriate verification activity? 

54 For certain products, moisture content, salt content and distribution, and the water droplet 

55 size/distribution are important for microbiological stability, therefore, testing for moisture, fat and salt 

56 serve as both quality and safety parameters; pH is an important parameter for testing in cultured butter 

57 and unsalted butter that is acidified as an additional barrier to microbial growth. If the temperature of 

58 incoming milk or cream is >45°F/7.2°C, load would typically be rejected rather than testing for enterotoxin. 

59 

60 Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

61 evidence that the appropriate treatment was, in fact, applied? 

62 Yes, if pasteurization and other process controls, as well as environmental control, are verified, 

63 finished product may not need microbial verification testing other than for quality as required by 

64 customers. However, if the product is open to cross-contamination, results from environmental 

65 monitoring will direct whether product testing is appropriate. 

66 
 

 

 

 

 

 

 

 

 

 

 

 

 

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67 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

68 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

69 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? What are 

70 appropriate indicator microorganisms for verifying processes adequately control pathogens? 

71 Because butter is made with pasteurized cream and may contain salt, routine product testing is 

72 limited to indicators of process control and sanitation (e.g., enumeration of Enterobacteriaceae or 

73 coliforms and aerobic plate count). Customer requirements, such as for butter produced in plants 

74 operating under USDA AMS inspection and grading services, may include additional quality-based microbial 

75 specifications (proteolytic count, not more than 100 CFU/g; yeast and mold count, not more than 20 CFU/g; 

76 coliform count, not more than 10 CFU/g, found in 7 CFR 58.345 and for whipped butter, proteolytic count, 

77 not more than 50 CFU/g; yeast and mold count, not more than 10 CFU/g; coliform count, not more than 

78 10 CFU/g; found in 7 CFR 58.346). 

79 

80 Question 5. What principles and criteria should a company apply in determining the frequency of testing 

81 finished product to determine if the company’s food safety system for that product is effective? 

82 Environmental exposure during processing, environmental monitoring program results, and ability 

83 to meet processing limits (such as pH, salt, or moisture as determined by the hazard assessment), should 

84 be used to determine type and frequency of finished product testing. In general, routine finished product 

85 testing for Enterobacteriaceae or coliforms (or other microbial targets identified by 7 CFR 58.345 or 7 CFR 

86 58.346) should be used to verify overall process control and sanitation. 

87 Several situations will indicate that additional product testing is necessary. For example, 

88 investigative testing may be needed when populations of indicator organisms exceed specified limits (e.g., 

89 >10 CFU/g coliforms or Enterobacteriaceae) suggesting insufficient sanitation, or if environmental 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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monitoring for Listeria spp. suggests that contamination by L. monocytogenes may have occurred during 

91 the production process (test for absence of L. monocytogenes; see product testing recommendations in 

92 (70)). Other investigative testing includes if there is a failure to achieve formulation parameters (such as 

93 insufficient acidification for unsalted or cultured butter) or processing time (such as extended interruptions 

94 during production) that may have allowed for growth of S. aureus; in such cases, test finished product to 

ensure <104 CFU/g S. aureus as appropriate. 

96 

97 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

98 the goal of verifying the adequacy of control of microbial hazards? 

99 Yes, testing aerobic colony count and Enterobacteriaceae or coliforms can be done on product 

obtained during production, as well as environmental testing. ATP detection is a useful and quick tool to 

101 verify that cleaning and sanitation removed organic matter off lines and product contact surfaces. 

102 

103 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

104 product testing verification activities by companies? 

Efficiency of cleaning should be verified for equipment and environment before process start-up, 

106 such as by visual inspection, ATP detection, or by testing aerobic colony count at an appropriate 

107 frequency. If microbial testing for indicator organisms exceeds limits, investigate source of 

108 contamination and implement corrective actions for sanitation and test final product to ensure 

109 corrections have been made. As an RTE product exposed to the environment, EMP for Listeria spp. 

should be implemented to reduce the need for testing finished product for L. monocytogenes. If 

111 EMP suggest that contamination of product by L. monocytogenes may have occurred, refer to 

112 guidance for testing product (68). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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113 

114 Question 8: What criteria should a company apply in determining that microbial testing results indicate 

a loss of (systemic) process control? What actions should a company take if test results indicate a loss 

116 of control? When verification testing indicates loss of process control, to what extent should verification 

117 testing be increased, how far upstream and downstream should it go, and when and how should it be 

118 scaled back? 

119 Table A-1 of this appendix, the Compliance Policy Guide for Dairy products (64) and the 

Department of Defense (50) outline microbial limits for various microbes and populations in finished 

121 product that can be used as verification of process control. If routine testing for indicators such as 

122 Enterobacteriaceae or coliforms in the finished product, or Listeria spp. in the environment, are out of 

123 specification and indicate loss of process control, facility should investigate source of failure to meet limits 

124 and implement corrective action; reference industry and government guidance documents for 

environmental testing for Listeria spp., product testing for L. monocytogenes, and follow-up testing needed 

126 to ensure process is back in control (30, 31, 63, 68). Products at risk of being contaminated with pathogens 

127 should be placed on hold and kept in the company’s control until follow-up testing is completed and lot is 

128 cleared. 

129 In addition, to microbial testing, other indicators of process control include monitoring of chemical 

and physical parameters of finished product (such as salt-in-moisture and pH) and production time131 temperature (such as extended runs or production down time). Delays during production or out of 

132 specification temperatures, particularly for unsalted butter with high water activity and high pH, could 

133 allow pathogen growth. Under these circumstances, investigative testing in implicated finished product 

134 (e.g., testing for Staphylococcus aureus) is recommended. 
 

 

 

 

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Page A-9 of 92 

136 

137 

138 

Table A-1. Microbial targets, limits, and recommended actions if limits are exceeded, for butter either 

refrigerated or formulated with sufficient salt or lactic acid to prevent growth; products containing added 

seasoning/herbs/spices may have additional requirements. 

Target Microorganism Microbial Limit Recommended Action if 

Limit is Exceeded 

Comments 

Coliforms or 

Enterobacteriaceae 

<10 CFU/g Investigate reason for 

exceeding limit and correct 

Routine testing 

Aerobic plate count 

(APC, SPC) 

<103 CFU/g Investigate reason for 

exceeding limit and correct 

This assay is not 

appropriate for 

cultured butter that 

will have higher 

counts due to use of 

starter culture 

Proteolytic <100 CFU/g 

<50 CFU/g 

whipped butter 

Investigate reason for 

exceeding limit and correct 

Testing for USDA 

AMS specifications, 

in 7 CFR 58.345, 7 

CFR 58.346 

Yeast and mold <20 CFU/g 

<10 CFU/g 

whipped 

Investigate reason for 

exceeding limit and correct 

Testing for USDA 

AMS specifications, 

in 7 CFR 58.345, 7 

CFR 58.346 
 

 

 

 

 

 

 

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Target Microorganism Microbial Limit Recommended Action if 

Limit is Exceeded 

Comments 

S. aureus <100 CFU/g Investigate, implement 

corrective action. 

If >104 CFU/g, reject lot due 

to potential for enterotoxin 

production 

Investigative testing 

if loss of process 

control is suspected. 

Listeria monocytogenes Absent in 25 g Destroy lot or divert to 

appropriate use with a 

lethality step. Investigate 

cause of contamination. 

Determine if other lots are 

involved. Determine steps to 

prevent reoccurrence. 

Investigative testing 

as response to EMP 

that suggests likely 

contamination of 

product 

139 

140 Recommendations for butter: 

141 • Because there is a pasteurization step for cream, no finished product testing for pathogens is 

142 needed when an effective EMP program is in place and other non-microbial monitoring 

143 (physiochemical and temperature/time controls) verifies that the process is in control. 

144 • Microbial testing of product should be focused on indicator organisms that reflect post-process 

145 contamination. 

146 o Enterobacteriaceae or coliforms populations should be <10 CFU/g. 
 

 

 

 

 

 

 

 

 

 

 

 

 

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o Plants operating under USDA AMS inspection and grading services may have additional 

customer requirements for aerobic colony counts, yeast/mold, and/or proteolytic 

microbes 

• In addition to microbial testing for indicator organisms, monitor physiochemical properties of the 

butter that are important in formation control of microbes (such as pH, salt-in-moisture, fat) and 

temperature-time for processing. 

• Microbial testing for pathogens in product is part of investigative actions (e.g., in response to out 

of compliance environmental results, inadequate formulation control, or inadequate 

temperature/time control), rather than routine testing. 

o If EMP suggest that contamination of product by L. monocytogenes may have occurred, 

refer to industry and government guidance documents for testing product. 

o If results for formulation parameters or time-temperatures controls indicate a loss of 

process control, enumeration S. aureus in product that exceeds 104 CFU/g will determine 

disposition of the product 

Cheese 

Cheese is made by coagulating milk with acid (developed by starter or direct acidification), acid in 

combination with heat, or rennet. Classifying cheese by texture (hard, grating, semi-soft, soft) is driven by 

moisture, with water holding capacity influenced by pH, pressing of curd, and aging. Both moisture and 

salt content impact water activity (aw), with aw 0.87 and 0.92 inhibitory to S. aureus enterotoxin production 

and L. monocytogenes growth, respectively, and aw <0.95 inhibitory to Salmonella and E. coli O157:H7. 

Cheese matrices may be inhibitory at higher water activities (e.g., >0.95) depending on product pH, acid 

type, presence of other competitive microbes, and antimicrobial compounds produced by starter or 
 

 

 

 

 

 

 

 

 

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169 adjunct cultures (3, 21, 42). However, pathogens can survive for extended months at reduced aw, 

170 depending on other stress conditions. 

171 While moisture is typically stable or decreases during ripening, the pH of cheeses frequently 

172 increases during aging due to growth of molds on surface-ripened and blue-veined cheeses or by 

173 deamination of proteins by starter and non-starter lactic acid bacteria proteolytic enzymes. For example, 

174 the pH of the curd immediately after pressing may be pH 5.2 but may increase to 5.8 or 7.0 in Gruyere and 

175 Camembert, respectively, after the ripening process. Because of the dynamic conditions for many cheeses, 

176 evaluating hazards for a given cheese should consider contaminating pathogens at the end of initial 

177 production (curd at pressing) and at packaging, as well as throughout aging and storage. 

178 

Classification Examples Typical Moisture ranges1 

Extra hard, grating Parmesan, Medium and Old Asiago <37% 

Hard Cheddar, Colby <40% 

Semi-soft Edam, Gouda, Brick, Muenster, Provolone, Blue, 

Low-Moisture Part Skim Mozzarella, Swiss 

40-50% 

Soft, ripened, Camembert, Brie, 50-60% 

Soft, unripened Queso fresco, Queso de Crema, Queso de Puna, 

fresh mozzarella, cream cheese, feta, ricotta 

>50% 

179 

180 A special note is for cheese made with unpasteurized milk because risks may be present in these 

181 cheeses that are not present in cheeses made from pasteurized milk. Specifically, cheeses made with 

1 While individual cheeses may have limits for moisture identified in CFR standards of identity, moisture within 

general classifications based on texture or ripening is not regulated. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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unpasteurized milk should consider additional testing of pathogens in finished product depending on 

thermization or other heat treatment, make procedures, cultures, product moisture, pH, brining, and aging 

conditions. Current regulations in the United States allow the sale of certain cheeses with standards of 

identity to be made from unpasteurized milk if the cheese is aged for at least 60 days at a minimum 

temperature of 35°F (1.7°C). However, aging may not be sufficient to eliminate low infectious dose 

pathogens, such as E. coli O157:H7 (18) and, depending on product moisture and pH, certain cheeses could 

allow growth of pathogens such as L. monocytogenes (26, 61) 

Cheese, hard (e.g., Cheddars), extra hard, grating (e.g., Parmesan, Romano) 

Microbiological safety issues are extremely rare in hard cheeses made with pasteurized milk and 

active starter cultures and adequate environmental controls. If cheese milk is contaminated (through use 

of unpasteurized milk or through post-pasteurization contamination in the vat), pathogens populations can 

increase/concentrate in the curd; Salmonella, STEC, and Listeria monocytogenes do not grow but can 

survive for several months during aging. Cheeses with slow starter activity (slow acidification) have been 

associated with growth of S. aureus and staphylococcal enterotoxin that can remain active during the aging 

process (76). 

Very hard cheeses, such as intact Parmesan and Romano, are inhibitory to growth of bacterial 

pathogens due to low aw (e.g., <0.92); validation studies have shown that these cheeses are typically nonTCS (non-time/temperature control for safety)(3, 21, 38, 42). Hard cheeses, such as Cheddar and Gruyere, 

with water activity <0.95, initial pH <5.6, and residual starter activity are similarly inhibitory to pathogens 

but are typically refrigerated for quality (3, 42). Low-salt hard cheeses that have water activity >0.95 and 

pH >5.6 may require temperature control to ensure stability unless validation studies suggest otherwise. 
 

 

 

 

 

 

 

 

 

 

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204 The presence of active cultures in hard cheeses makes the use of routine microbiological testing 

205 for aerobic plate count (known as APC or SPC) impractical as a tool for evaluation of process controls and 

206 sanitary conditions. In contrast, testing for coliforms or Enterobacteriaceae, which are destroyed by 

207 pasteurization, can serve as an indication of post-process contamination from insanitary conditions for 

208 cheeses made with pasteurized milk. Testing for S. aureus or generic E. coli is useful under special 

209 circumstances such as investigation when production has occurred without adequate process control. For 

210 cheeses made with unpasteurized milk, additional pathogen testing is recommended both for the milk as 

211 well as for finished product. For all cheeses, regardless of the use of pasteurized milk, regular 

212 environmental testing of the food production environment for the presence of Listeria spp. and Salmonella 

213 is recommended as a verification step for sanitation programs. 

214 

215 Example 1 - Extra hard cheese made with pasteurized milk (e.g., Parmesan, Asiago for grating) finished 

216 product aw typically <0.91 

217 Example 2 - Hard cheese made with unpasteurized milk (e.g., raw milk Cheddar); finished product aw 

218 typically <0.95 

219 

220 Question 1. What principles and criteria should a company apply in determining the need for and in 

221 designing an effective microbial testing program to verify that processes are effectively controlling 

222 microbial pathogens? 

223 
 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Extra hard cheese made with 

pasteurized milk (e.g., Parmesan, 

Asiago for grating) finished product 

aw typically <0.91 

Hard cheese made with 

unpasteurized milk (e.g., raw milk 

Cheddar); finished product aw 

typically <0.95 

A. Are pathogens No, pasteurization of milk used for Yes, raw milk may contain multiple 

associated with the cheesemaking will eliminate pathogens, including Salmonella, E. 

food or ingredients? vegetative bacterial pathogens, 

rendering it safe for use. Food 

safety issues for this cheese type 

are rare. 

Use of adjunct ingredients, such as 

fresh herbs or spice rub, could serve 

as a source of contamination. 

coli O157:H7 and other STEC, L. 

monocytogenes, S. aureus, and 

Brucella. Outbreaks have been 

associated with raw milk cheeses 

due to survival of low infectious 

dose pathogens beyond 60-day 

aging (16, 29) 

B. Are the ingredients Hard cheeses made with Surveys suggest 1-2% of raw milk 

likely to be pasteurized milk are rarely samples used for artisan cheeses 

contaminated? contaminated. Ingredients such as 

spices and herbs, the environment, 

or food handlers may be a source of 

Salmonella, E. coli O157:H7, L. 

monocytogenes or S. aureus. 

contain one or more pathogens 

(17). Bulk milk samples can have 

higher rates of contamination (58). 

C. Are there robust 

processing control 

procedures such as a 

Milk pasteurization is a robust kill 

step for most vegetative pathogens. 

Mild heat treatments, such as 

thermization may reduce 

pathogens by only 1 or 2 logs, 
 

 

 

 

 

 

 

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Criterion/Factor Extra hard cheese made with 

pasteurized milk (e.g., Parmesan, 

Asiago for grating) finished product 

aw typically <0.91 

Hard cheese made with 

unpasteurized milk (e.g., raw milk 

Cheddar); finished product aw 

typically <0.95 

kill step or other 

reduction 

methods/controls? 

Suppliers should have a validated 

lethality treatment for 

ingredients/inclusions (spices, 

herbs, fruits, etc.) added post 

lethality. Sanitation will reduce 

microbes on food contact surfaces 

and in the environment. 

unless temperature-time 

combinations been validated for 

efficacy. The aging/ripening process 

for hard and very hard cheeses will 

reduce pathogen load over time, 

but 60-day aging may be 

insufficient to qualify as a robust 

reduction step in raw milk cheeses. 

Rate of inactivation relies on 

combined stresses such as drying 

(low water activity), acidity, residual 

starter activity, and storage 

temperatures >3°C to accelerate 

lethality during aging. Although the 

aging process of cheeses inactivates 

pathogens over time, some low 

infectious dose pathogens, such as 

E. coli O157:H7, have been shown 

to survive months. Sanitation will 
 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Extra hard cheese made with 

pasteurized milk (e.g., Parmesan, 

Asiago for grating) finished product 

aw typically <0.91 

Hard cheese made with 

unpasteurized milk (e.g., raw milk 

Cheddar); finished product aw 

typically <0.95 

reduce microbes on food contact 

surfaces and in the environment. 

D.Is there a potential Yes, there can be potential for Yes, there can be potential for 

for recontamination recontamination of the cheese milk cross-contamination of the cheese 

from the handling or during curd development and milk during curd development and 

the environment? handling, brining, during the aging 

process, portioning, or packaging. 

handling, brining, during the aging 

process, portioning, or packaging. 

E. Does the product 

support survival or 

growth? 

Most extra hard cheeses (finished 

product) do not support growth of 

pathogens during aging and storage 

due to combinations of reduced 

water activity, pH/acidity, and 

residual starter culture activity. 

If acidification rate is compromised 

due to slow or failed starter culture 

activity during the cheesemaking 

process, pathogens such as S. 

aureus may grow and produce 

enterotoxin. While populations of 

Most hard cheeses (finished 

product) do not support growth of 

pathogens during aging and storage 

due to combinations of reduced 

water activity, pH/acidity, and 

residual starter culture activity. 

These stresses will typically result in 

inactivation of pathogens during 

aging, but hard cheeses made with 

contaminated milk may have 

pathogen survival for >60 days. 
 

 

 

 

 

 

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Criterion/Factor Extra hard cheese made with 

pasteurized milk (e.g., Parmesan, 

Asiago for grating) finished product 

aw typically <0.91 

Hard cheese made with 

unpasteurized milk (e.g., raw milk 

Cheddar); finished product aw 

typically <0.95 

vegetative cells will decline during 

aging of the cheese, enterotoxin 

will be stable. 

If acidification rate is compromised 

due to slow or failed starter culture 

activity during the cheesemaking 

process, pathogens such as S. 

aureus may grow and produce 

enterotoxin. While populations of 

vegetative cells will decline during 

aging of the cheese, enterotoxin 

will be stable. 

F. Is this product meant This food is not specifically This food is not specifically 

for higher risk intended for high-risk populations, intended for high-risk populations, 

population? but people from high-risk 

populations may choose to 

consume this type of product 

but people from high-risk 

populations may choose to 

consume this type of product 

G.What is the shelf life 

of the product? 

Variable, several years. Variable, several years. 
 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Extra hard cheese made with 

pasteurized milk (e.g., Parmesan, 

Asiago for grating) finished product 

aw typically <0.91 

Hard cheese made with 

unpasteurized milk (e.g., raw milk 

Cheddar); finished product aw 

typically <0.95 

H.Will consumer 

handling and use 

increase or decrease 

likelihood of 

pathogen survival, 

growth, or toxin 

production and risk 

of consumer illness? 

Because many cheeses within these 

categories are non-TCS, 

temperature abuse by the retailer 

or consumer or holding food 

beyond use-by date will have little 

impact on pathogen survival, 

growth, or toxin production and 

hence no increased risk for 

consumer illness. 

Because many cheeses within these 

categories are non-TCS, 

temperature abuse by the retailer 

or consumer or holding food 

beyond use-by date will have little 

impact on pathogen survival, 

growth, or toxin production and 

hence no increased risk for 

consumer illness. 

224 

225 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

226 enzymes, would be an appropriate verification activity? 

227 Although alkaline phosphatase (ALP) can serve as an indicator of pasteurization, residual heat in 

228 very large wheels of raw milk hard cheese can lead to inactivation of ALP (52).. Pathogen growth is 

229 inhibited in milk during cheese making by robust starter culture activity and acid development. Therefore, 

230 monitoring pH during acidification of the curd will detect slow fermentation that may result in a product 

231 that can support the growth of pathogens. Testing in-process or finished product for moisture, salt and/or 

232 water activity is important to verify formulation control. Testing for alkaline phosphatase can serve as an 

233 indicator of pasteurization (63). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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234 

Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

236 evidence that the appropriate treatment was, in fact, applied? 

237 When the process is in control, microbial testing of indicator organisms is an appropriate 

238 verification activity because there are several points in the manufacturing process where post239 pasteurization contamination could occur (curd development, pressing, brining, packaging, conversion to 

slices/shreds, etc.). 

241 

242 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

243 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

244 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

246 In general, intact hard and extra hard cheeses are unlikely to support the growth of pathogens due 

247 to a combination of suboptimal water activity, total acidity, and competitive microbiota. A company should 

248 use published or commissioned validation studies or use “safe harbors” such as aw or pH growth limits (32, 

249 67, 68) to identify pathogen growth inhibition parameters for specific cheese types, particularly for nonrefrigerated conditions. Under these situations, routine enumeration of indicator organisms in pasteurized 

251 milk, along with determining that the growth inhibition parameters are met for the cheese, may be 

252 sufficient for verification of process control. 

253 Converting cheese into sliced, shredded, or grated forms may disrupt the inherent safety system. 

254 In general, finished product testing is not an effective verification tool in hard cheeses due to the 

distribution and low levels of potential contaminants. However, finished product testing may be 

256 appropriate in cases where the converted cheese (such as slices for prepared refrigerated sandwiches, 
 

 

 

 

 

 

 

 

 

 

 

 

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262

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264

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275

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pieces for deli salads, etc.) could potentially deliver contaminants, such as L. monocytogenes, in foods that 

could support growth of pathogens (e.g., test presence/absence L. monocytogenes in 25 g samples). 

Regardless of product composition, aging and storage, environmental monitoring for Listeria spp. 

and Salmonella (68) and routine enumeration for Enterobacteriaceae or coliforms (<100 CFU/g) in finished 

product can be used as indicators of post-process contamination. If acid production is slow, it is advisable 

to test for coagulase-positive staphylococci or S. aureus (<104 CFU/g) 

Cheeses made with raw or thermized milk are more likely to have pathogens derived from the milk 

that have survived during aging; therefore, testing finished product for presence/absence of E. coli 

O157:H7 and/or Salmonella can be used as verification that preventive controls have been adequately 

implemented. 

Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

Because of the great diversity of cheese types produced in many regions, as well as production, 

consumption, and distribution practices, it is difficult to recommend specific universally applicable testing 

for all cheese types. Testing frequency will be facility and line dependent, and should consider the 

• Potential for recontamination (such as sanitation, extended runs, brine management, etc.) 

• Intrinsic properties of the cheese (pH, water activity, microbiota, validation studies that identify 

pathogen growth, survival, or die-off), aging/ripening conditions, product history of recalls or 

related illness 

• Intended use (retail, food service, institutions) 

• Facility-specific process; extent of exposure and handling post-lethality prior to packaging 
 

 

 

 

 

 

 

 

 

 

 

 

 

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290

295

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279 • Facility-specific infrastructure condition (floors, walls, ceilings, separation of raw/RTE, traffic 

control, etc.) 

281 • Facility-specific sanitary condition of equipment/processing lines 

282 • Internal testing history (product and environmental) 

283 

284 For example, for cheeses that do not support growth of L. monocytogenes (61) and in facilities with a 

robust environmental monitoring program, routine testing (such as on a lot basis) for indicators of post286 process contamination and sanitation, such as Enterobacteriaceae, may be more effective to verify that 

287 the food safety system is in control than finished product testing. For cheeses made with raw milk, having 

288 a COA or testing incoming milk for Salmonella and Listeria can provide insight for likelihood of 

289 contamination of the finished cheese after minimum aging. If cheese is positive for pathogens at any point 

during aging, reject the lot unless cheese can be reconditioned to eliminate the pathogen. 

291 

292 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

293 the goal of verifying the adequacy of control of microbial hazards? 

294 Environmental monitoring and in-process testing (e.g., in curd after pressing) before aging provides 

more useful information to evaluate the safety of the product. Monitoring the pH of curd can detect slow 

296 fermentation and indicate that testing for S. aureus (<104 CFU/g) may be relevant if acidification does not 

297 proceed as anticipated. Testing for indicator organisms (e.g., molds, yeasts, Enterobacteriaceae, or 

298 Listeria-like microorganisms) in brine or curd for E. coli (<100 CFU/g) in cheese made from heat-treated 

299 milk may be useful to verify process control and hygiene conditions. For raw milk cheese, pull milk samples 

from the cheese vat after a homogeneous mix or utilize a disposable filter sock on the milk discharge pipe 

301 to the vat, after fill. In addition, supplier control of milk can be achieved through herd management, 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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302 mastitis control, focus on feeding regimens, and sanitation during milking, storage and transportation to 

303 the cheese makers (2). Microorganisms are likely to decline during the aging process of hard and very hard 

304 cheeses; in particular for raw milk cheeses that rely on the aging process to control microbial hazards, 

305 testing finished product for pathogens will serve as a final verification that process was effective before 

306 release of the product. 

307 

308 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

309 product testing verification activities by companies? 

310 Hard cheeses do not support growth of pathogens, but routine environmental monitoring for 

311 Listeria species (Listeria-like microorganisms as an indicator for L. monocytogenes) and Enterobacteriaceae 

312 (as an indicator of sanitation), and/or Salmonella can determine if post-process contamination problem 

313 spots exist (30, 31, 68, 75). If environmental monitoring identifies positive samples, follow guidelines for 

314 corrective action and resampling (30, 68). Cheese that has been made from raw milk should be tested for 

315 Listeria spp., E. coli (STEC) and Salmonella at 60-day age prior to release for distribution (2). 

316 

317 Question 8: What criteria should a company apply in determining that microbial testing results indicate 

318 a loss of (systemic) process control? 

319 What actions should a company take if test results indicate a loss of control? 

320 When verification testing indicates loss of process control, to what extent should verification testing be 

321 increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

322 The Compliance Policy Guide for Dairy products (64) and Department of Defense (50) outlines 

323 microbial limits for various pathogens and recommendations for indicator organisms. A facility should 

324 track/trend test results (using a 3-class sampling plan for indicators such as E. coli, Enterobacteriaceae, 
 

 

 

 

 

 

 

 

 

 

 

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325 etc.) and have identified action limits e.g., for pass, warning, and fail. Reacting to results trending up, in the 

326 warning level, will prevent a loss of control. In addition, a company should reference federal and industry 

327 guidance documents for environmental testing for Listeria (30, 31, 63, 68). 

328 

329 When there is a loss of systemic process control, there should be a root cause analysis performed, 

330 including looking at sanitation procedures, environmental controls, heat-treatment of milk, supplier 

331 controls (such as for inclusions, rubs, surface cultures, or raw milk), cheese vat/make procedures, 

332 acidification rate, finishing table, brine tanks, block formation, aging, cutting, and packaging to determine 

333 the source(s) of loss of control and implement corrective action. The findings will dictate the extent to 

334 which verification testing is increased. 

335 

336 Table A-2. Microbial targets, limits, and recommended actions if limits are exceeded, for hard cheeses 

337 made with pasteurized milk. Additional testing may be indicated for cheeses made with raw milk where 

338 low infectious dose pathogens, such as E. coli O157:H7 may survive 60-day aging requirements (2). 

Target 

Microorganism 

Microbial Limit Recommended Action if Limit is 

Exceeded 

Comments 

Coliforms or 

Enterobacteriaceae 

<100 CFU/g Investigate reason for exceeding 

limit and implement corrective 

action; consider testing for E. coli 

(<10 CFU/g) if coliforms are 

detected 

Routine testing 

S. aureus <100 CFU/g Investigate reason for exceeding 

limit and implement corrective 

Investigative testing for S. 

aureus if pH monitoring of 
 

 

 

 

 

 

 

 

 

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action. If >104 CFU/g, reject lot due 

to potential for enterotoxin 

production (64). Due to heat 

stability of enterotoxin, diverting 

to further processing is not 

recommended 

a vat suggests acid 

development is slow. 

Investigate reason for slow 

pH drop, implement 

corrective action. 

Listeria Absent in 25 g Reject lot. Investigate cause of Investigative testing as 

monocytogenes contamination. Determine if other 

lots are involved. Determine steps 

to prevent reoccurrence. 

response to EMP that 

suggests likely 

contamination of product 

Salmonella Negative in 375 

g analytical 

units (15 x 25-g 

samples) 

Reject lot. Testing may be inprocess vat sample due to the 

aging process for natural cheese 

Investigative testing as 

response to EMP that 

suggests likely 

contamination of product 

or routine testing for 

cheeses made with raw 

milk and aged for 60 days. 

339 

340 Recommendations for hard, extra hard, and grating cheese. 

341 • For hard and very hard cheeses made with pasteurized milk and that are known to inhibit growth 

342 of pathogens during normal storage, no routine finished product testing for pathogens is needed 

343 when an effective EMP program is in place and other non-microbial monitoring (such as monitoring 

344 acid development) suggests process is in control. 
 

 

 

 

 

 

 

 

 

 

 

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350

355

360

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• Routine enumeration testing of product for indicator organisms of adequate sanitation (e.g., 

346 Enterobacteriaceae or coliforms) and environmental monitoring for Listeria spp. should be 

347 conducted to identify loss of process control and to identify risks of post-process contamination 

348 (30, 68). 

349 • Investigative microbial testing of finished products for pathogens (e.g., L. monocytogenes or 

Salmonella) should be conducted if other testing suggests inadequate environment control and 

351 likely product contamination. 

352 o If EMP suggest that contamination of product by L. monocytogenes or Salmonella may 

353 have occurred, refer to industry and government guidance documents for testing product. 

354 • If monitoring of acidification rate suggests poor starter activity, which can allow growth of 

infectious and toxigenic microorganisms enumeration S. aureus, investigative testing of pressed 

356 curd for S. aureus should be conducted. 

357 o Destroy lots where enumeration of S. aureus in product exceeds 104 CFU/g 

358 • In addition to above, for hard and very hard cheeses made with unpasteurized milk (2) testing of 

359 milk supply and finished product for pathogens with low infectious dose and long survival times, 

such as E. coli O157:H7 and Salmonella, is appropriate to verify that thermization and ripening 

361 processes are sufficient to eliminate these pathogens. 

362 • Microbiological testing of finished product for pathogens is also recommended if an ingredient that 

363 has a potential to contain pathogens such as herbs, spices, or other inclusions, are added post364 pasteurization; ingredients added after pasteurization should be obtained from approved suppliers 

and subjected to supplier verification activities. Ingredients from a new supplier with little history 

366 may require addition verification testing 

367 
 

 

 

 

 

 

 

 

 

 

 

 

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368 Cheese (soft, semi-soft, surface ripened) 

369 Examples: fresh (Queso fresco), soft, soft-ripened (Camembert), semi-soft (Edam, Gouda), 

veined cheeses (Roquefort, Gorgonzola) 

371 

372 This category represents a broad range of cheeses. Contaminated soft and semi-soft (ripened and 

373 unripened) cheeses made with pasteurized or raw milk have been associated with listeriosis; illnesses from 

374 Salmonella and shiga-toxin producing E. coli have been attributed to consumption of semi-soft cheeses 

made with raw milk. 

376 Routine environmental monitoring for Listeria spp. in the environment and coliforms in finished 

377 product should occur for all products in this category to identify loss of process control. However, for 

378 higher pH (>pH 5.4) soft cheeses known to support the growth of L. monocytogenes and that have been 

379 implicated in illness, use of pasteurized milk, and aggressive environmental controls and monitoring, as 

well as periodic finished product testing for indicators and pathogens is appropriate (30, 56, 61, 68). 

381 Testing for S. aureus and generic E. coli may be used when processing or insanitary conditions indicate a 

382 potential increased microbiological risk. For cheeses made with unpasteurized milk, testing milk will 

383 provide insights for likelihood of residual pathogens that may be present after aging. 

384 

Example 1 - Cheese, fresh (Queso fresco), typical aw >0.98 

386 Example 2 - Cheese, semi-soft, Gouda made with unpasteurized milk; typical aw 0.96-0.98 

387 

388 Question 1. What principles and criteria should a company apply in determining the need for and in 

389 designing an effective microbial testing program to verify that processes are effectively controlling 

microbial pathogens? 
 

 

 

 

 

 

 

 

 

 

 

 

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Page A-28 of 92 

391 

392 

393 

Principles that apply to finished product testing of this RTE food: 

Examples 

Criterion/Factor Cheese, fresh (Queso fresco) 

Typical aw >0.98 

Cheese, semi-soft, Gouda made 

with unpasteurized milk; typical aw 

0.96-0.98 

A. Are pathogens Pasteurization of milk used for Raw milk may contain multiple 

associated with the cheesemaking will eliminate pathogens, including Salmonella, E. 

food or ingredients? vegetative bacterial pathogens, 

rendering it safe for use. However, 

recontamination of curd/finished 

cheese from the environment or by 

contaminated adjunct ingredients 

has been associated with outbreaks 

from Salmonella, STEC, and Listeria 

monocytogenes. Fresh (such as 

queso fresco) and soft, surfaceripened cheese with high pH (>5.2) 

are known to support growth of L. 

monocytogenes, whereas soft 

cheeses with pH <5.2 (lactic acid 

predominant) are typically 

coli O157:H7, L. monocytogenes, S. 

aureus, and Brucella. If cheese milk 

is contaminated, microbial 

populations can 

increase/concentrate in the curd. 

Gouda made with unpasteurized 

milk has been associated with long 

survival of E. coli O157:H7 and with 

illness. 60 days aging may be 

insufficient to inactivate low 

infectious dose pathogens. Cheeses 

with slow starter activity (slow 

acidification) have been associated 

with growth of S. aureus; and 
 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Cheese, fresh (Queso fresco) 

Typical aw >0.98 

Cheese, semi-soft, Gouda made 

with unpasteurized milk; typical aw 

0.96-0.98 

inhibitory to pathogens when 

refrigerated. 

staphylococcal enterotoxin that can 

remain active. 

B. Are the ingredients Pasteurized milk is rarely Surveys suggest 1-2% of raw milk 

likely to be contaminated, but curd can be samples used for artisan cheeses 

contaminated? recontaminated during the make 

procedure, brining, or packaging. 

Adjunct ingredients such as spices 

and herbs or the environment may 

be a source of Salmonella or L. 

monocytogenes. 

contain one or more pathogen(17). 

Bulk milk samples can have higher 

rates of contamination (58). 

C. Are there robust 

processing control 

procedures such as a 

kill step or other 

reduction 

methods/controls? 

Legal pasteurization is sufficient kill 

step for vegetative pathogens. 

If curd is recontaminated, 

pathogens are likely to survive or 

grow during storage, depending on 

product pH. 

Unless validated for lethality, mild 

heat treatments such as 

thermization may reduce 

pathogens by as little as 1 or 2 logs 

(target suggested to be 3-log 

reduction). 

The aging/ripening process for 

semi-soft cheeses can reduce 

pathogen load, but 60-day aging 

may be insufficient to qualify as a 
 

 

 

 

 

 

 

 

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Criterion/Factor Cheese, fresh (Queso fresco) 

Typical aw >0.98 

Cheese, semi-soft, Gouda made 

with unpasteurized milk; typical aw 

0.96-0.98 

robust reduction step. Rate of 

inactivation relies on combined 

stresses such as drying, acidity, and 

residual starter activity and storage 

temperatures >3°C to accelerate 

lethality during aging. Some low 

infectious dose pathogens, such as 

E. coli O157:H7, have been shown 

to survive months. 

D.Is there a potential Yes, there can be potential for Yes, there can be potential for 

for recontamination recontamination during the curd recontamination during production, 

from the handling or stage or packaging. aging, portioning, or packaging. 

the environment? 

E. Does the product 

support survival or 

growth? 

Yes, L. monocytogenes grows in soft 

cheeses, particularly with pH >5.2 

during normal refrigerated storage. 

Refrigeration will inhibit growth of 

mesophilic pathogens, but survival 

times may be months. 

Gouda does not support pathogen 

growth due to low initial pH (<5.3) 

and levels of undissociated lactic 

acid (74), but low infectious dose 

pathogens such as E. coli O157:H7 

have been shown to survive for 

months during aging (18). 
 

 

 

 

 

 

 

 

 

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Criterion/Factor Cheese, fresh (Queso fresco) 

Typical aw >0.98 

Cheese, semi-soft, Gouda made 

with unpasteurized milk; typical aw 

0.96-0.98 

F. Is this product meant This food is not specifically This food is not specifically 

for higher risk intended for high-risk populations, intended for high-risk populations, 

population? but people from high-risk 

populations may choose to 

consume this type of product.” 

but people from high-risk 

populations may choose to 

consume this type of product.” 

G.What is the shelf life 

of the product? 

Typically, 60-90 days Gouda may be aged from 60 days to 

several years 

Will consumer handling Storage at temperatures >3°C Storage temperature and time that 

and use increase or increases the risk of L. exceed marked conditions are 

decrease likelihood of monocytogenes growth in fresh and unlikely to affect safety if 

pathogen survival, certain other soft cheeses with composition is inhibitory to 

growth, or toxin pH>5.2, depending on predominant pathogens; however, quality will be 

production and risk of acid. Slight temperature abuse or diminished. Validation studies for 

consumer illness? extended storage times can 

increase the risk of growth and 

consumer illness. Other mesophilic 

pathogens will survive, but not 

grow, in these cheeses held at <7°C. 

specific formulation may be 

necessary to determine risks if held 

at temperatures greater than 4°C 

394 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

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Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

enzymes, would be an appropriate verification activity? 

Testing for alkaline phosphatase can serve as an indicator of pasteurization of milk used for making 

cheese (63). Testing in process or finished product for moisture, salt and/or water activity is important to 

verify formulation control. Pathogen growth is inhibited in milk during cheese making by rapid acidification. 

Therefore, monitoring formulations (such as volume of acid per volume of milk for direct acid set 

production) and monitoring pH during acidification of the curd will detect inadequate 

acidification/fermentation that may result in an unstable product. 

Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

evidence that the appropriate treatment was, in fact, applied? 

No, because there are several points in the manufacturing process where post-pasteurization 

contamination could occur. Microbial testing is an appropriate verification activity. 

Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

A company should consider the potential of individual cheeses to support growth of pathogens, 

under refrigerated and non-refrigerated conditions, if it were contaminated. Validation of growth 

inhibition for specific cheese types, particularly for non-refrigerated conditions, is useful in determining 

potential risks and therefore, pathogens of concern. 
 

 

 

 

 

 

 

 

 

 

 

 

 

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417 Regardless of product composition, aging and storage, environmental monitoring for Salmonella 

418 and Listeria species (68) and routine testing of finished product for Enterobacteriaceae or coliforms (<100 

419 CFU/g) can be used as indicators of post-process contamination. If acid production is slow, it is advisable 

420 to test for coagulase positive staphylococci or S. aureus (<104 CFU/g). Cheeses made with raw milk should 

421 consider additional pathogen testing of the final product depending on results for raw milk, environmental 

422 positive tests, and levels of indicator organisms. Soft cheeses made with pasteurized milk should also be 

423 considered for finished product pathogen testing because of the risk of cross-contamination post kill step, 

424 the ability of pathogens to grow in the product, and the history of safety issues with this product type. 

425 Converting cheese into slice, shred, or grated forms may disrupt the inherent safety system. In 

426 addition, the end use of the cheese (such as slices used as an ingredient for prepared refrigerated 

427 sandwiches, pieces for deli salads, etc.) needs to consider the potential for delivering contaminants, such 

428 as L. monocytogenes, to foods that may support growth of pathogens or spoilage microbes. In this case, 

429 finished product testing for L. monocytogenes may be indicated. 

430 

431 Question 5. What principles and criteria should a company apply in determining the frequency of testing 

432 finished product to determine if the company’s food safety system for that product is effective? 

433 Because of the great diversity of cheese types produced in many regions; as well as production, 

434 consumption, and distribution practices, it is difficult to recommend specific universal applicable testing 

435 for all cheese types. For example, for cheeses that support growth of L. monocytogenes, finished product 

436 should routinely be tested for indicator organisms and for L. monocytogenes. For cheeses that are made 

437 with pasteurized milk and do not support pathogen growth, greater reliance on indicator organism testing 

438 and environmental monitoring should be sufficient to assure process control. 

439 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

440

445

450

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Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

441 the goal of verifying the adequacy of control of microbial hazards? 

442 Environmental monitoring provides useful information to determine the potential for post443 pasteurization contamination. Monitoring the pH of curd can detect slow fermentation and testing for S. 

444 aureus (<104 CFU/g) may be relevant if acidification does not proceed as anticipated. Testing for indicator 

organisms (e.g., molds, yeasts, Enterobacteriaceae, or Listeria-like microorganisms) in brine or curd for E. 

446 coli (<100 CFU/g) in cheese made from heat-treated milk is useful to verify process control and hygiene 

447 conditions. For raw milk cheese, pull milk samples from the cheese vat after a homogeneous mix or utilize 

448 a disposable filter sock on the milk discharge pipe to the vat, after fill. In addition, supplier control of milk 

449 can be achieved through herd management, mastitis control, focus on feeding regimens, and sanitation 

during milking, storage and transportation to the cheese makers (2). 

451 

452 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

453 product testing verification activities by companies? 

454 Testing for Listeria spp. in processing environments is important to verify the effectiveness of 

implemented hygiene control measures; verification of rigorous environmental controls can be justification 

456 for reduced finished product testing. Frequency and extent of product testing verification should be based 

457 on history and established criteria. Refer to guidance documents for frequency and locations for 

458 environmental monitoring (30, 31, 68). 

459 

Question 8: What criteria should a company apply in determining that microbial testing results indicate 

461 a loss of (systemic) process control? 

462 What actions should a company take if test results indicate a loss of control? 
 

 

 

 

 

 

 

 

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463 When verification testing indicates loss of process control, to what extent should verification testing be 

464 increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

465 Refer to sources such as the PMO, federal and industry guidance documents, and Department of 

466 Defense microbiological limits for indicators of loss of process control and actions to be taken if results 

467 indicate a loss of process control (30, 31, 50, 63, 68). 

468 When there is a loss of systemic process control, there should be a root cause analysis performed, 

469 including heat-treatment of milk, supplier controls (such as for inclusions or raw milk), cheese vat/make 

470 procedures, acidification rate, finishing table, brine tanks, block formation, aging, cutting, and packaging 

471 to determine the source(s) of loss and implement corrective action. The findings will dictate increases in 

472 verification testing. 

473 

474 Table A-3. Microbial targets, limits, and recommended actions if limits are exceeded, for soft cheeses made 

475 with pasteurized milk. Additional testing may be indicated for cheeses made with raw milk (2, 26). 

Target 

Microorganism 

Microbial 

Limit 

Recommended Action if Limit is 

Exceeded 

Comments 

Coliforms or 

Enterobacteriaceae 

<100 CFU/g Investigate reason for exceeding 

limit and implement corrective 

action; consider testing for E. coli 

(10 CFU/g) if coliforms are 

detected 

Routine testing 

S. aureus <100 CFU/g Investigate reason for exceeding 

limit and implement corrective 

action. If >104 CFU/g, reject lot 

Investigative testing for S. 

aureus if pH monitoring of 

a vat suggests acid 
 

 

 

 

 

 

 

 

 

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due to potential for enterotoxin development is slow. 

production. Due to heat stability Investigate reason for 

of enterotoxin, diverting to further slow pH drop, implement 

processing is not recommended corrective action 

Listeria Negative in Reject lot. Investigate cause of Investigative testing as 

monocytogenes 125 g 

analytical 

units (5 x 25-

g samples) 

contamination. Determine if other 

lots are involved. Determine steps 

to prevent reoccurrence. 

response to EMP that 

suggests likely 

contamination of product 

or routine testing for 

products that can support 

growth of L. 

monocytogenes 

Salmonella Negative in 

375 g 

analytical 

units (15 x 25 

g samples) 

Reject lot. Investigate cause of 

contamination. Determine if other 

lots are involved. Implement 

corrective action to prevent 

reoccurrence. 

Investigative testing as 

response to EMP that 

suggests likely 

contamination of product 

or routine testing for 

cheeses made with raw 

milk and aged for 60 days 

476 

477 Recommendations for soft cheese. 

478 • Routine testing of product for indicator organisms (e.g., Enterobacteriaceae) and environmental 

479 monitoring for Listeria spp. (30, 68) are recommended to identify loss of process control. 
 

 

 

 

 

 

 

 

 

 

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480 • Investigative finished product testing for pathogens (e.g., L. monocytogenes or Salmonella) should 

481 be conducted if testing suggests inadequate environment control and likely product 

482 contamination. 

483 o If EMP suggest that contamination of product by L. monocytogenes or Salmonella may 

484 have occurred, refer to industry and government guidance documents for testing product. 

485 • Additionally, routine testing (e.g., daily, weekly, or quarterly depending on facility) of finished 

486 product for L. monocytogenes may be appropriate for products that can support growth of this 

487 pathogen during shelf-life. 

488 • If monitoring of acidification rate suggests poor starter activity which can allow growth of 

489 infectious and toxigenic microorganisms enumeration S. aureus, investigative testing of pressed 

490 curd for S. aureus should be conducted. 

491 o Destroy lots where enumeration of S. aureus in product exceeds 104 CFU/g 

492 • Microbiological testing of finished product for pathogens is also recommended if an ingredient that 

493 has a potential to contain pathogens such as herbs, spices, or other inclusions, are added post494 pasteurization. 

495 o Ingredients added after pasteurization should be obtained from approved suppliers and 

496 subjected to supplier verification activities. 

497 o Ingredients from a new supplier with little history may require addition verification testing 

498 • Finished cheeses made with raw milk should also consider routine testing of pathogens such as L. 

499 monocytogenes, Salmonella, and E. coli O157:H7 in raw milk supply and finished product (2) 
 

 

 

 

 

 

 

 

 

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Cultured, pH < 4.8 

501 Examples include buttermilk, sour cream, kefir, koumiss, and yogurts. These products are 

502 inherently safe due to pasteurization of the milk to eliminate vegetative pathogens, robust fermentation 

503 (lactic acid production) to prevent growth and enterotoxin production and use of qualified ingredients used 

504 as inclusions. These finished products do not support growth of pathogens, and with sufficient lactic acid 

present they can inactivate vegetative pathogens during storage. 

506 Briefly, the products described in this section are produced by fermenting milk at mild 

507 temperatures (e.g., 110-115°F) to convert lactose to lactic acid by the metabolism of the specific beneficial 

508 bacterial cultures that are added for preservation and flavor. The production of the lactic acid reduces the 

509 pH of the milk to the isoelectric point of casein (pH 4.6), but curd will be formed at slightly higher pH of 

4.8. The time to achieve a pH <4.8 can range between 4 and 12 hours (>12h for kefir), depending on 

511 fermentation temperature and cultures used. Robust fermentation has been shown to outcompete 

512 various pathogens, including E. coli O157:H7, L. monocytogenes, Salmonella, Staphylococcus aureus, and 

513 Bacillus cereus, particularly when pH is reduced to <5.0 with lactic acid (12, 22, 48, 57). Products are filled 

514 into cups and chilled and stored refrigerated for the duration of shelf-life. 

Monitoring fermentation rate is the primary indicator that the fermentation is inhibitory to 

516 pathogens. Furthermore, products with pH <4.8 (where lactic acid is predominant) have been shown to 

517 not support pathogen growth and will slowly inactivate pathogens during storage. If the rate of pH 

518 decrease is compromised, testing for pathogens should be considered. The product may be exposed to 

519 the environment during preparation and filling of containers and contaminants could be introduced by 

additions after pasteurization (such as fruit puree, caramel or chocolate sauce, nuts, etc.). 
 

 

 

 

 

 

 

 

 

 

 

 

 

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521 Question 1. What principles and criteria should a company apply in determining the need for and in 

522 designing an effective microbial testing program to verify that processes are effectively controlling 

523 microbial pathogens? 

524 

Criterion/Factor Cultured Dairy, pH <4.8 

A. Are pathogens associated 

with the food or 

ingredients? 

Although raw milk and inclusions used in cultured dairy products can 

contain pathogens, cultured dairy products made with pasteurized 

milk and rapidly fermented to pH <4.8 (where lactic acid is 

predominant) are rarely associated with illness. Sporeformers such as 

B. cereus or C. perfringens will survive pasteurization; however, 

germination and outgrowth are controlled through fermentative 

acidification that produces a rapid pH drop below levels that permit 

growth. Acid tolerant pathogens such as E. coli O157:H7 will have the 

longest survival time but will not grow. Fermentation and resulting 

acid production are control measures, but producer should be alert to 

delay of acid development if starter cultures are compromised, 

such as due to presence of inhibitory substances such as antibiotics 

or phages. 

B. Are the ingredients likely Pasteurized milk is unlikely to be contaminated. Recontamination 

to be contaminated? can occur through the addition of ingredients such as fruit 

concentrates or pulps, pastes, or syrups, chocolate, nuts, or other 

inclusions. Ingredients that have been heat treated and acidified have 

a low probability of pathogens, but may include yeasts and molds, 
 

 

 

 

 

 

 

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Criterion/Factor Cultured Dairy, pH <4.8 

which may change the product pH over time. Supplier verification for 

these ingredients is advised. Additionally, starter cultures should 

meet specifications, including lack of phage contamination. 

C. Are there robust 

processing control 

procedures such as a kill 

step or other reduction 

methods/controls? 

Yes. Milk pasteurization is a validated reduction method. The acid 

environment (<4.8, lactic acid) will continue to reduce pathogens over 

time, but reduction may be slow (e.g., days or weeks at 4°C). 

D.Is there a potential for 

recontamination from 

the handling or the 

environment? 

Yes. Given the nature of the processing environment, which are 

frequently wet cleaned, potential for recontamination would most 

likely be from spoilage microorganisms. 

E. Does the product support 

survival or growth? 

No. The acidity of the product (<4.8, lactic acid) does not support 

growth of bacterial pathogens and will limit their survival. The acid 

environment will continue to reduce pathogens over time, but 

reduction will be slow. However, acid tolerant spoilage bacteria, 

yeasts or molds may grow. 

F. Is this product meant for 

higher risk population? 

High-risk populations may consume these foods, but food is not 

specifically intended for this demographic. 
 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Cultured Dairy, pH <4.8 

G.What is the shelf life of 

the product? 

4-6 weeks, depending on the product 

H.Will consumer handling 

and use increase or 

decrease likelihood of 

pathogen survival, 

growth, or toxin 

production and risk of 

consumer illness? 

Extended refrigeration will not affect safety due to the low pH of the 

product. Holding cultured dairy products at temperatures greater 

than 4°C will accelerate acid development, which will affect quality 

but will not compromise safety. 

525 

526 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

527 enzymes, would be an appropriate verification activity? 

528 Yes. pH testing during fermentation is essential and should be done continuously or at regular 

529 intervals to verify fermentation is sufficiently robust as to prevent growth of pathogens in the milk. 

530 

531 Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

532 evidence that the appropriate treatment was, in fact, applied? 

533 If records show that the acidification rate to <4.8 was rapid (e.g., within 5 hours or per validation 

534 study for given product) and EMP verifies sanitation, no pathogen testing for the white mass is needed. 

535 Microbial testing for indicator organisms for sanitation and spoilage organisms is recommended 

536 particularly if inclusions/adjunct ingredients are used. 

537 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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538 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

539 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

541 What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

542 Because product is made with pasteurized milk and robust fermentation, pathogen testing of 

543 product is not needed unless acid development is slow or environmental monitoring program indicate loss 

544 of process control or sanitation failure. Enumeration testing for indicators of sanitation for postpasteurization environment (e.g., Enterobacteriaceae or coliforms) or for spoilage microorganisms such as 

546 molds, yeasts or gas-producing lactic acid bacteria, e.g., Leuconostoc spp., is useful as a check for supplier 

547 control of adjunct ingredients/inclusions. 

548 

549 Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

551 Ability to meet specifications for acid development, environmental exposure during processing, 

552 environmental monitoring program results, and supplier control for adjunct ingredients/inclusions that are 

553 added after fermentation, should be used to determine type and frequency of finished product testing. In 

554 general, routine finished product testing for Enterobacteriaceae, or coliforms or spoilage/mold/yeasts 

should be used to verify overall process control and sanitation. End product testing for pathogens is not 

556 routinely conducted because monitoring of the fermentation in-process provides the most actionable 

557 information. 

558 Ingredients and inclusions (such as fruit puree, caramel or chocolate sauce, nuts, etc.) added after 

559 pasteurization should be obtained from approved suppliers and subjected to supplier verification activities. 

Ingredients from a new supplier with little history may require addition verification testing. 
 

 

 

 

 

 

 

 

 

 

 

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Several situations will indicate that additional product testing is necessary. For example, 

investigative testing for pathogens should be conducted if slow acid development suggests the potential 

for pathogen growth in the product if it were contaminated by L. monocytogenes or S. aureus from the 

environment or employees during the production process. Investigative testing is needed when 

populations of indicator organisms exceed specified limits suggesting insufficient sanitation or inadequate 

supplier control for incoming ingredients used for inclusions. If environmental monitoring for Listeria 

spp. suggests that contamination by L. monocytogenes may have occurred during the production process 

(test for absence of L. monocytogenes; see product testing recommendations in U.S. Food and Drug 

Administration, 2017 (69)). 

Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

the goal of verifying the adequacy of control of microbial hazards? 

Because acid development is the primary preventive control for cultured products made with 

pasteurized milk, non-microbial testing should include monitoring the pH of the white mass during 

fermentation routinely to detect slow fermentation. Environmental monitoring provides useful 

information to determine the potential for post-pasteurization contamination. Because niches and 

sanitation requirements may be different for gram negative microbes (e.g., Enterobacteriaceae) compared 

to gram positive bacteria and fungi, samples for testing of gas-forming lactic acid bacteria, yeasts, and 

molds are best taken from the adjunct ingredients or from critical pieces of equipment such as 

intermediate storage tanks, balance tanks, fillers, etc. to verify no buildup of these microorganisms before 

starting each day’s production. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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583 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

584 product testing verification activities by companies? 

A robust environmental testing program for indicator and spoilage microorganisms will reduce the 

586 need for finished product testing. When determining disposition of cultured product where acid 

587 production was delayed, results that demonstrate Listeria is under control will reduce the need to test for 

588 the pathogen in finished product; follow recommendations for testing outlined for cultured dairy products 

589 with pH >4.8 but <5.2 and industry and government guidance (30, 31, 68). The EMP should also periodically 

test for Salmonella, which is associated with low moisture ingredients such as dairy powders, nuts, or other 

591 adjunct ingredients and could become established in the environment. Verifying that this pathogen is 

592 controlled in the environment reduces the risk that it will be a post-process contaminant in the finished 

593 product. 

594 

Question 8: What criteria should a company apply in determining that microbial testing results indicate 

596 a loss of (systemic) process control?

597 What actions should a company take if test results indicate a loss of control?

598 When verification testing indicates loss of process control, to what extent should verification testing be 

599 increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

When acid production fails, revealing loss of systemic process control, the company should initially 

601 investigate the milk product for inhibitory substances to fermentation. If investigative pathogen testing 

602 reveals the potential of heat-stable enterotoxin production produced by S. aureus (such as >104 CFU/g of 

603 S. aureus), lot should be discarded. If populations of S. aureus are below the threshold for enterotoxin 

604 production, further review of EM results for Listeria spp. and potentially finished product testing for L. 

monocytogenes should be considered (see recommendations outlined for cultured dairy products with pH 
 

 

 

 

 

 

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606 >4.8 but <5.2 for testing). When cause of loss of process control has been corrected, routine verification 

607 testing can be resumed. 

608 

609 Table A-4. Microbial targets, limits, and recommended actions if limits are exceeded, for cultured dairy 

610 products made with pasteurized milk (e.g., Sour cream, yogurt, buttermilk) and active pH control. 

Target 

Microorganism 

Microbial 

Limit 

Recommended Action if Limit is 

Exceeded 

Comments 

Coliforms or 

Enterobacteriaceae 

<10 CFU/g Investigate reason for exceeding 

limit and implement corrective 

action; consider testing for E. coli 

if coliforms are detected at >10 

CFU/g. 

Routine testing, 

particularly for products 

with ingredient additions 

after fermentation is 

complete 

Mold/Yeast <10 CFU/g Investigate, implement corrective 

action 

Testing routinely is 

dependent on the product 

if no mold inhibitor is 

added. The presence of 

mold and yeast may be 

influenced by added 

ingredients such as fruit 

purees and other 

inclusions. Fungal growth 

on the surface of a 

product can increase the 
 

 

 

 

 

 

 

 

 

 

 

 

 

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pH and disrupt the 

formulation safety based 

on pH. 

S. aureus <100 CFU/g Investigate reason for exceeding 

limit and implement corrective 

action. If >104 CFU/g, reject lot 

due to potential for enterotoxin 

production. Due to heat stability 

of enterotoxin, diverting to further 

processing is not recommended 

Investigative testing for S. 

aureus if fermentation 

does not reach pH <4.8 in 

<5 h (or other rate 

determined by challenge 

study data). Investigate 

reason for slow pH drop, 

implement corrective 

action 

611 

612 Recommendations for testing cultured dairy products with pH < 4.8: 

613 • Routine product testing is for rate of acid development. 

614 • Environmental monitoring and finished product for indicator organisms is a verification of process 

615 control, supplier control, and sanitation. 

616 • Pathogen testing of product is not recommended unless acid development is slow, which indicates 

617 loss of process control 

618 • Ingredients and inclusions added after pasteurization should be obtained from approved suppliers 

619 and subjected to supplier verification activities. 

620 o Ingredients from a new supplier with little history may require addition verification testing 

621 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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622 Cultured, pH > 4.8 and <5.4 

623 Examples include acidophilus milk, hot-filled or cold-filled cream cheese or cottage cheese. 

624 Cultured dairy products are inherently safe due to pasteurization of the milk to eliminate vegetative 

625 pathogens; robust fermentation with lactic acid producing bacteria prevents pathogen growth during the 

626 culturing process through acid development and competition with the culture. However, additions of 

627 cream or other adjunct ingredients may raise the pH of the finished product that may affect growth 

628 potential of pathogens and spoilage microbes. 

629 Briefly, products are manufactured using fluid milk or cream heated to 90o

C for several minutes to 

630 remove competing bacteria. Pasteurized milk or cream is cultured with a lactic acid bacteria starter culture, 

631 frequently to pH 4.8. The addition of the adjunct ingredients (such as cream dressing, vegetable material, 

632 salmon, or other inclusions) can raise the equilibrated pH to values between 4.8 and 5.4. While hot filling 

633 for certain cream cheese and cottage cheese products eliminates concerns for vegetative pathogens and 

634 spoilage microbes, sporeforming bacteria can survive. Although pH 4.6 is considered the limit to prevent 

635 growth of sporeformers at room temperature, cultured dairy products with pH <5.4 using lactic acid will 

636 slow growth (extend lag and generation times) and therefore product can tolerate limited extended cooling 

637 (challenge study data is required to validate that cooling profile is sufficient to inhibit spore outgrowth for 

638 product of concern). However, other pathogens, such as L. monocytogenes can potentially grow in these 

639 products if present, even during refrigerated storage, unless formulation adjustments have been made. 

640 

641 Question 1. What principles and criteria should a company apply in determining the need for and in 

642 designing an effective microbial testing program to verify that processes are effectively controlling 

643 microbial pathogens? 

644 
 

 

 

 

 

 

 

 

 

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Criterion/Factor Cultured, equilibrated pH > 4.8 and <5.4 

A. Are pathogens associated 

with the food or 

ingredients? 

Although raw milk can contain pathogens, products are made with 

pasteurized milk or cream are rarely associated with illness. 

Sporeformers such as B. cereus or C. perfringens may survive 

pasteurization; however, germination and outgrowth are controlled 

through fermentation or direct acidification that produces a rapid pH 

drop below levels that permit growth. On rare occasion, the presence 

of inhibitory substances such as antibiotics or phages can delay acid 

production, allowing growth of pathogens if present. 

B. Are the ingredients likely Pasteurized milk and cream are unlikely to be contaminated with 

to be contaminated? vegetative pathogens, but spores can survive heat. Recontamination 

from the environment can occur, particularly during addition of 

adjunct ingredients to the curd. Ingredients that may be cold blended 

into the curd such canned smoked salmon, pasteurized fruit 

concentrates or pulps, heat treated pastes or syrups, nuts, chocolate, 

brined peppers, dehydrated carrots, dried chives, or natural and 

artificial flavors are infrequently contaminated, but supplier 

verification is advised. Additionally, starter cultures should meet 

specifications, including lack of phage contamination. 

C. Are there robust Yes. Milk and cream are heated to pasteurization or ultraprocessing control pasteurization temperatures to eliminate vegetative pathogens and 

procedures such as a kill other microbes that may interfere with the culturing process. 

Surviving spore outgrowth is slowed by competition with starter 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Cultured, equilibrated pH > 4.8 and <5.4 

step or other reduction 

methods/controls? 

culture and by rapid lactic acid development to reduce pH to <5.2. 

Hot-filling products, such as for some types of cream cheese or 

cottage cheese products, will eliminate vegetative pathogens but will 

allow survival of spores. 

D.Is there a potential for 

recontamination from 

the handling or the 

environment? 

Yes. Given the nature of the processing environments, which are 

frequently wet cleaned, potential for recontamination would be from 

spoilage microorganisms or L. monocytogenes. Addition of 

ingredients post fermentation process or cold filling into packaging 

can introduce contaminants. 

E. Does the product support 

survival or growth? 

Growth of pathogens is inhibited by a combination of pH <5.0 with 

lactic acid, temperature-control, and/or addition of synthetic or clean 

label antimicrobials. Products with pH >5.0 and no antimicrobials 

have potential to support growth of L. monocytogenes even with 

refrigerated temperature control. 

F. Is this product meant for 

higher risk population? 

High-risk populations may consume these foods, but food is not 

specifically intended for this demographic. 

G.What is the shelf life of 

the product? 

60-90 days 

H.Will consumer handling 

and use increase or 

decrease likelihood of 

pathogen survival, 

For intact packages of hot-filled products, such as brick cream cheese, 

extended refrigerated holding beyond use-by date is unlikely to 

increase risk. However, cold-filled product without preservatives, 

such as certain cottage cheese products with pH >5.0, can support 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Cultured, equilibrated pH > 4.8 and <5.4 

growth, or toxin 

production and risk of 

consumer illness? 

growth of L. monocytogenes; extended refrigerated storage beyond 

use-by date or holding at temperatures greater than 4°C can increase 

the risk that food may support growth of L. monocytogenes and 

increase the risk of illness. 

645 

646 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

647 enzymes, would be an appropriate verification activity? 

648 Yes. pH testing during culturing of the milk at regular intervals is essential to ensure robust 

649 fermentation and adequate pathogen control. 

650 

651 Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

652 evidence that the appropriate treatment was, in fact, applied? 

653 Yes. Microbial verification testing is not necessary for hot-filled products (with a validated thermal 

654 process) that have had a robust fermentation and have met cooling requirements. 

655 However, for products that are cold filled and therefore are exposed to the environment or via 

656 addition of ingredients, testing for indicator organisms should be conducted in addition to the 

657 environmental monitoring and supplier control programs. 

658 

659 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

660 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

661 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

662 What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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For products that are not hot filled, enumeration of Enterobacteriaceae or coliforms (e.g., less than 

10 CFU CFU/g) can be used as an indicator of post-pasteurization contamination. For products that also 

have inclusions/adjunct ingredients, consider the recommendations for appropriate target microbes and 

microbiological limits in the appendices of this document for those ingredients. For example, while the 

limit for coliforms is <10 CFU CFU/g in dried chives or spice blends, acceptable populations of 

Enterobacteriaceae is 102 CFU CFU/g; therefore, when added to cold-blended cream cheese, the final 

populations of Enterobacteriaceae may exceed 10 CFU/g and use of coliforms may be a better indicator 

organism. 

The presence of molds and yeasts may be influenced by added ingredients such as fruit purees and 

dehydrated vegetables or herbs. For products that do not contain antimycotic agents, testing for molds 

(<10 CFU/g) may be necessary because mold growth on the product surface can increase the pH of the 

product, disrupting the safety system established by the reduced pH. 

Environmental testing of Listeria species, Salmonella is recommended for products that are not 

hot filled; additional testing for mold/yeasts (air) are useful for facilities with products that do not contain 

mold growth inhibitors or packaging that excludes mold growth. 

Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

Ability to meet specifications for acid development, environmental exposure during processing, 

temperature at which product is filled, environmental monitoring program results, and supplier control for 

adjunct ingredients/inclusions that are added after fermentation, should be used to determine type and 

frequency of finished product testing. 
 

 

 

 

 

 

 

 

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705

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Products that have a validated lethality step and hot fill do not require microbial testing for finished 

686 product. For products that are filled at temperatures are lower than that needed for lethality, but in 

687 facilities with a robust environmental control and supplier control programs, pathogen testing is not 

688 routinely conducted but routine finished product testing for indicators of sanitation (e.g., 

689 Enterobacteriaceae or coliforms) or spoilage/mold/yeasts should be used to verify overall process control 

and sanitation. 

691 Several situations will indicate that additional product testing is necessary. For example, 

692 investigative testing for pathogens (e.g., S. aureus and/or L. monocytogenes) should be conducted if slow 

693 acid development suggests the potential for pathogen growth in the product. Investigative testing is 

694 needed when environmental monitoring for Listeria spp. or populations of indicator organisms in finished 

product exceed specified limits suggesting insufficient sanitation or inadequate supplier control for 

696 incoming ingredients used for inclusions. 

697 

698 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

699 the goal of verifying the adequacy of control of microbial hazards? 

In addition to microbial testing at the end of process, pH testing of the milk/cream during 

701 fermentation to monitor acid production should be done routinely to ensure adequate acid production to 

702 control microbial hazards. Samples for testing of spoilage microbes as indicators of sanitation (e.g., 

703 heterofermentative gas-forming lactic acid bacteria, yeasts, and molds) can be taken from critical pieces 

704 of equipment such as intermediate storage tanks, balance tanks, fillers, etc., particularly during extended 

runs. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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706 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

707 product testing verification activities by companies? 

708 A robust environmental monitoring program that demonstrates that Listeria (and molds, as 

709 relevant) are in control will reduce the need for finished product testing because they are microorganisms 

710 of concern in cultured dairy products with pH >4.8. The EMP should also periodically test for Salmonella, 

711 which is associated with dairy powders, dehydrated vegetables, herbs, and spices, or other low moisture 

712 ingredients, and which could contaminate and become established in the environment. Verifying that this 

713 pathogen is controlled in the environment reduces the risk that it will be a post-process contaminant in 

714 the finished product 

715 If results from environmental monitoring program suggests potential for contamination of the 

716 finished product, it could result in the increased need for microbiological testing of product or ingredients 

717 for L. monocytogenes as part of investigative testing or root cause analysis (30, 31, 68). 

718 

719 Question 8: What criteria should a company apply in determining that microbial testing results indicate 

720 a loss of (systemic) process control?

721 What actions should a company take if test results indicate a loss of control?

722 When verification testing indicates loss of process control, to what extent should verification testing be 

723 increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

724 Because cold-filled cultured dairy products are expected to contain high populations of starter 

725 culture, testing the environment and monitoring other process controls (e.g., acid production and cooling 

726 rates), are more actionable tests of loss of process control. When acid production is slow or stalls or cooling 

727 deviations occur revealing loss of systemic process control, the company should initially investigate causes. 

728 Product should be placed on hold and evaluated to ensure there was no potential for growth of toxigenic 
 

 

 

 

 

 

 

 

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729 pathogens to levels that could cause illness. Follow FDA Draft Guidance on environmental monitoring to 

730 verify control of L. monocytogenes (68) 

731 

732 Table A-5. Microbial targets, limits, and recommended actions if limits are exceeded, for cultured dairy 

733 products with pasteurized milk, pH >4.8 and < 5.4, moisture >50%; active pH control required (Ex. Cottage 

734 cheese, cream cheese) 

Target 

Microorganism 

Microbial Limit Recommended Action if 

Limit is Exceeded 

Comments 

Coliforms or 

Enterobacteriaceae 

(EB) 

<10 CFU/g Investigate reason for 

exceeding limit and 

implement corrective action. 

If >10 CFU/g and regulated 

under PMO, reject lot due to 

regulatory limit 

Routine testing. However, 

products that have added 

dried herbs and vegetables 

may have populations of 

Enterobacteriaceae that 

are higher than 10 CFU/g. 

See Appendix F 

Spices/Herbs for guidance 

for testing. 

S. aureus <100 CFU/g Investigate reason for 

exceeding limit and 

implement corrective action. 

Investigate, implement 

corrective action. 

Investigative testing for S. 

aureus if fermentation is 

slow where the pH of the 

curd does not reach pH 

<4.8 in <8 h (or other rate 

depending on challenge 
 

 

 

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Target 

Microorganism 

Microbial Limit Recommended Action if 

Limit is Exceeded 

Comments 

if >104 CFU/g, reject lot due 

to potential for enterotoxin 

production 

study data). Investigate 

reason for slow pH drop, 

implement corrective 

action. 

Molds <10 CFU/g Investigate, implement 

corrective action 

Testing is dependent on 

the product. Mold can 

raise the pH of the product, 

disrupting the safety 

system of the product. 

Considerations are the type 

of inclusion/ingredient, 

whether product is hot 

filled, has effective 

antimycotic agents added, 

or packaging excludes 

oxygen to inhibit molds 

during storage. 

735 
 

 

 

 

 

 

 

 

 

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736 Recommendations for testing cultured dairy products with pH > 4.8 and <5.2: 

737 • Routine product testing for acid development 

738 • Routine testing for indicator organisms (e.g., coliforms or Enterobacteriaceae, and molds as 

739 appropriate) 

740 • Environmental monitoring programs (e.g., for Listeria spp., Salmonella and molds as appropriate) 

741 as verification of sanitation 

742 • Pathogen testing of product is not recommended unless environmental monitoring program 

743 suggests risk of post-pasteurization contamination or acid development is slow, which suggests 

744 loss of process control 

745 • Ingredients and inclusions added after pasteurization should be obtained from approved suppliers 

746 and subjected to supplier verification activities. 

747 o Ingredients from a new supplier with little history may require addition verification testing 

748 
 

 

 

 

 

 

 

 

 

 

 

 

 

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749 Dried Dairy Products 

750 Examples include dry milk powder (e.g., non-fat dried milk), lactose, whey products, buttermilk 

751 powder, dried cheese powder, and infant formula (21 CFR part 106). Pasteurization of the fluid milk will 

752 kill vegetative pathogens, but spores will survive, including throughout the drying process. Whey derived 

753 from cheese making may be stored in silos prior to processing (fractionation, concentration, concentration, 

754 drying), with temperature/time limits to prevent growth of bacteria such as S. aureus and B. cereus that 

755 can produce heat stable enterotoxin; these temperature/time limits should be included in the food safety 

756 plan. Dried dairy products are common ingredients in products that will not receive a kill step prior to 

757 consumption such as chocolate, cold pressed energy bars and energy drink powders that are rehydrated 

758 with cold water. Salmonella and L. monocytogenes are potential hazards for these products, with 

759 additional concerns for ingredients used in infant formula including Cronobacter spp. 

760 

761 Question 1. What principles and criteria should a company apply in determining the need for and in 

762 designing an effective microbial testing program to verify that processes are effectively controlling 

763 microbial pathogens? 

Criterion/Factor Dried Dairy Products 

A. Are pathogens associated 

with the food or 

ingredients? 

Epidemiological data suggest that Salmonella is a significant hazard 

that needs to be controlled during the manufacture of dried dairy 

products intended for consumption by all populations. Outbreaks of 

Salmonella have been reported in dried milk and infant formula (11, 

14, 54, 73). S. aureus and B. cereus can be present in dairy powders 

and can present an issue if the product is reconstituted and abused. 

(36). Cronobacter and Clostridium botulinum are hazards in products 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Dried Dairy Products 

for infants, although testing specifically for C. botulinum is not useful 

as a process control (5, 34, 65). Listeria monocytogenes is a hazard for 

dried milk products that have RTE uses. 

B. Are the ingredients likely Ingredients such as caseinates, whey powder, and other milk 

to be contaminated? derivatives, vitamins, trace elements and minerals or lecithin may be 

added during processing. Certain ingredients, such as milk 

derivatives, have a known history of presence of Salmonella. While 

ingredients added before the heat treatment, (pasteurization) do not 

represent an issue, those added after the kill step represent a risk and 

therefore need to fulfill the same microbiological requirements as the 

finished product. Products are dried after milk pasteurization and 

thus may be contaminated from the environment. 

C. Are there robust Yes. Pasteurization of the fluid milk would precede spray drying. 

processing control However, the drying process itself (evaporation and spray/roller 

procedures such as a kill drying) is not considered a pathogen kill step, as Salmonella, L. 

step or other reduction monocytogenes, and Cronobacter can survive drying. Spores will 

methods/controls? survive both pasteurization and spray drying (59) 

D. Is there a potential for Yes, Salmonella contamination from the environment is a concern, as 

recontamination from is L. monocytogenes for RTE dried milk products. Cronobacter 

the handling or the contamination from the environment is a concern in products 

environment? intended for infants. Increased levels of Enterobacteriaceae in 

finished products can be used as an indicator of recontamination 
 

 

 

 

 

 

 

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Criterion/Factor Dried Dairy Products 

from the processing environment even though when Cronobacter 

populations are low, Enterobacteriaceae may be undetectable (15, 

20). 

Other hazards such as S. aureus or B. cereus (or the presence of 

preformed staphylococcal enterotoxins) are normally only present at 

very low levels and do not pose a risk as long as the products are not 

mishandled prior to pasteurization or after reconstitution and before 

consumption. Mishandling (holding time and temperature) would 

allow growth and toxin formation. 

E. Does the product support 

survival or growth? 

Dry product (aw 0.3-0.4) does not support microbial growth, but 

Salmonella and Cronobacter survive for extremely long periods 

(months). 

F. Is this product meant for 

higher risk population? 

Yes, if used in infant formula. 

G. What is the shelf life of 

the product? 

Extended shelf life (months to years) at room temperature. 

H. Will consumer handling 

and use increase or 

decrease likelihood of 

pathogen survival, 

growth, or toxin 

None of these is likely; however, the product can be rehydrated when 

used by the consumer. Pathogens such as Cronobacter and 

Salmonella can grow in the rehydrated product if they are present 

and temperature abuse occurs. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Dried Dairy Products 

production and risk of 

consumer illness? 

764 

765 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

766 enzymes, would be an appropriate verification activity? 

767 No. Testing for indicator organisms and/or pathogens is the appropriate verification activity. 

768 

769 Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

770 evidence that the appropriate treatment was, in fact, applied? 

771 Yes, for example, when the dried dairy product is used exclusively as an ingredient in foods that 

772 receive a validated lethality treatment. 

773 

774 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

775 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

776 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

777 What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

778 Enterobacteriaceae (enumeration) and Salmonella (absence) are the specific indicator and 

779 pathogen, respectively, used for testing in dried dairy products (35). In addition, a consideration for testing 

780 finished product is whether it is intended for high-risk populations, such as infants, the elderly, or 

781 immunocompromised individuals. In that instance, Cronobacter spp. is added to finished product testing 

782 (Cronobacter is mainly a concern in infants less than 12 months old)(13, 35). For ingredients used in infant 

783 formula, enumeration of mesophilic spores or sulfite-reducing Clostridia are used frequently in the industry 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

785

790

795

800

805

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784 as an indicator of process hygiene because spores can survive pasteurization and can be concentrated in 

dry ingredients (34). 

786 

787 Question 5. What principles and criteria should a company apply in determining the frequency of testing 

788 finished product to determine if the company’s food safety system for that product is effective? 

789 Given the history of contamination associated with dry dairy products and the difficulty in 

eliminating pathogens from the processing environment using dry cleaning methods, testing frequency for 

791 this commodity typically is greater than other dairy products described in this appendix. For products that 

792 have received a lethality step (e.g., initial milk pasteurization), increased levels of Enterobacteriaceae in 

793 finished products indicates recontamination from the processing environment. Because Salmonella falls 

794 within the family of Enterobacteriaceae, testing for this group of organisms can be performed as a hygiene 

indicator in the environment (23). However, while there is a correlation between finding 

796 Enterobacteriaceae and other gram-negative pathogens, Enterobacteriaceae is not always detected when 

797 Salmonella or Cronobacter are present in low numbers (6, 20, 33). 

798 Listeria monocytogenes is a hazard for dried milk products which are included in RTE products; 

799 therefore, environmental monitoring should be conducted to identify food surface, packaging, or other 

potential environmental contamination especially when a listericidal control is not consistently applied for 

801 final product testing (68). 

802 For manufacturers that test both in-process and environmental samples, a low frequency of end -

803 product testing for Salmonella is performed as a verification for a low number of samples for end-product 

804 testing. Depending on the product use and customer requirements, Salmonella testing in finished product 

may be conducted on each lot pre-shipment. The frequency of finished product testing will be impacted 

806 by the results of the environmental monitoring program, as well as the hygienic design of the line, the 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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807 ability to exclude water in sensitive processing areas, length of time for dryer or evaporator run, and the 

808 addition of ingredients after pasteurization. Note that customers who use dried milk products as 

809 ingredients in RTE foods may require testing of each lot for pathogens such as Salmonella. 

810 There are microbial specifications for Grade A dry milk powder, whey and buttermilk produced 

811 under the PMO, as well as dairy powders eligible for USDA grading or inspection services. Manufacturers 

812 of infant formula are required to perform testing for Salmonella and Cronobacter spp. on each production 

813 aggregate per 21 CFR 106.55. Although correlation between infant botulism and dried infant formula is not 

814 well established, testing for sulfite-reducing Clostridium spores is frequently used as an indicator of process 

815 hygiene for these ingredients (5, 34, 39). 

816 

817 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

818 the goal of verifying the adequacy of control of microbial hazards? 

819 In-process sampling and testing can confirm effective control measures. Sampling plans should 

820 include representative samples taken after the drying step through the filling operation; automatic samples 

821 are often used in dry milk filling operations. Product contact surfaces where residues accumulate could 

822 indicate areas of moisture condensation, and thus potential for microbial growth. Sampling points include 

823 sifter tailings from the after dryer/after cooler or from tipping stations of intermediate products and filling 

824 machines. 

825 

826 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

827 product testing verification activities by companies? 

828 Since the major cause of presence of Salmonella or increased levels of Enterobacteriaceae in 

829 finished products is recontamination from the processing environment, sampling and testing of 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

846

847

848

849

850

851

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environmental samples plays a key role in verifying the effectiveness of the preventive measures. One 

recommendation is testing Enterobacteriaceae in dry processing areas, such that target levels is <100 

CFU/swab and the action level is >1000 CFU/swab, depending on the proximity to product and product risk 

level (31). It should be noted that testing for Enterobacteriaceae alone is not suitable, since even low levels 

do not necessarily guarantee the absence of the pathogen. Frequency and extent of product testing should 

be increased if the results from environmental monitoring show the presence of Salmonella, or increased 

levels of EB, or if product is intended for immunocompromised individuals. 

Question 8: What criteria should a company apply in determining that microbial testing results indicate 

a loss of (systemic) process control? 

What actions should a company take if test results indicate a loss of control? 

When verification testing indicates loss of process control, to what extent should verification testing be 

increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

The limits for non-pathogenic indicator organisms listed in this document are intended to provide 

guidance for acceptable limits where little information is available for a process or product. However, it is 

expected that each facility will collect and analyze quantitative data to establish statistical process control. 

A loss of systemic process control is indicated when indicator data repeatedly exceed the limits established 

for a stable process operating within predictable process variation, or exceptionally high indicator levels 

are observed pathogens. Loss of control also includes detecting Salmonella, L. monocytogenes and 

Cronobacter (for infant formula) in finished or in-process product. 

When a loss of process control is indicated, the implementation and efficacy of preventive controls 

should be investigated before and after the drying process. These include sanitation, temperature and/or 
 

 

 

 

 

 

 

 

 

 

 

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852 hygiene segregation controls prior to spray drying fluid pasteurized product and sanitation, water and 

853 hygiene segregation controls after the spray drying process. 

854 When a pathogen is detected, the recommended action is to reject the lot of dried product 

855 represented by the sample tested and any contiguous runs not separated by a clean sanitation break. Due 

856 to lengthy process runs, special consideration needs to be given to lot definition and the establishment of 

857 a clean break if the product is contaminated with a pathogen such as Salmonella. The repeated finding of 

858 indicator organisms such as coliforms or Enterobacteriaceae above a threshold level can also indicate a 

859 loss of sanitation control although actions taken would follow a tiered approach based on numbers and 

860 frequency of occurrence. 

861 Finished product testing for Salmonella using FDA category II sampling is typically used on a per lot 

862 basis to screen for contamination (66). FDA Category II includes foods that would not normally be 

863 subjected to a process lethal to Salmonella between the time of sampling and consumption. The 

864 parameters of Category II are: 30 analytical units/ 25 g samples. The samples may be aggregated into 375 

865 g analytical units. If the ingredient is intended for a high-risk consumer (e.g., infants), testing of 60 X 25 g 

866 samples should be considered (see Category I food classification on Sample Schedule Chart 1 of 

867 Investigations Operations Manual (66). Product for L. monocytogenes testing is sampled in 25 g aliquots. 

868 Multiple 25 g samples are taken over a production lot and may be composited into a 125 g sample if this 

869 sample size is validated for the matrix. Dried milk products are sampled for Cronobacter in 10g increments 

870 over the course of a production run and may be composited into a 300 g sample. If any product tests 

871 positive for Salmonella or other pathogens, it should not be released for consumption, regardless of results 

872 from follow-up testing. 
 

 

 

 

 

 

 

 

 

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873 Many manufacturers use autosamplers to incrementally take small samples of dry material 

874 throughout a production run prior to packaging. These aggregated samples are tested in composites 

875 sample sizes (i.e., 375, 125 or 300 grams) appropriate for the pathogen analyzed. 

876 Control of toxigenic microorganisms such as S. aureus or B. cereus is limited to investigative testing 

877 if hold temperature and time exceed limits for liquid in-process milk products prior to drying identified in 

878 a food safety plan. 

879 

880 Table A-6. Microbial targets, limits, and recommended actions if limits are exceeded, for Dairy-Dried 

881 Products (Ex. NFDM, whey powder, including dried dairy ingredients used in infant formula). 

Target Microorganism Microbial Limit Recommended 

Action if Limit is 

Exceeded 

Comments 

Aerobic plate count 

(APC, SPC) 

<104 CFU/g Investigate, 

implement corrective 

action 

Routine testing. Acceptable 

aerobic plate count 

populations can be set by 

critical evaluation of trends 

for process control for 

individual line and facility 

Coliforms or 

Enterobacteriaceae 

<10 CFU/g Investigate, 

implement corrective 

action 

Routine testing. 

Salmonella Negative in 375 g Divert for 

reprocessing, if 

Routine testing. As an 

alternative sampling option 
 

 

 

 

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Target Microorganism Microbial Limit Recommended 

Action if Limit is 

Exceeded 

Comments 

appropriate, or 

destroy. Investigate 

and implement 

corrective action 

to collecting and compositing 

15-25 g samples (total 375 g), 

an auto sampler can be used 

to collect small amounts of 

samples throughout a 

production run for a total of 

375g; Recommend 1500 g 

per lot when high volumes of 

product are produced per lot 

(or production day). 

L. monocytogenes Negative in 25 g Divert for 

reprocessing, if 

appropriate, or 

destroy. Investigate 

and implement 

corrective action 

Investigative testing as 

response to EMP that 

suggests likely contamination 

of product. 

S. aureus <100 CFU/g Investigate, 

implement corrective 

action. 

Investigative testing, such as 

if hold temperature/time of 

components before drying 
 

 

 

 

 

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Target Microorganism Microbial Limit Recommended 

Action if Limit is 

Exceeded 

Comments 

if >102 CFU/g, reject 

lot due to potential 

for enterotoxin 

production 

exceed limits identified in 

food safety plan 

B. cereus <100 CFU/g Investigate, 

implement corrective 

action. 

if >104 CFU/g, reject 

lot due to potential 

for enterotoxin 

production 

Investigative testing, such as 

if hold temperature/time of 

components before drying 

exceed limits identified in 

food safety plan 

Testing is more stringent for ingredients used in infant formula (21 CFR 106.55) (34); 

Testing in addition to those described above 

Cronobacter Negative in 300 g; 

Composite sample 

30 samples x 10 g 

Divert for 

reprocessing, if 

appropriate, or 

reject. Investigate 

and implement 

corrective action 

Routine testing. 
 

 

 

 

 

 

 

 

 

 

 

 

 

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Target Microorganism Microbial Limit Recommended Comments 

Action if Limit is 

Exceeded 

Mesophilic spores or 

Sulfite-reducing 

Clostridium spores 

<100 spores/g Divert for 

reprocessing or 

alternate use if 

appropriate or reject. 

Investigate and 

implement corrective 

action 

Routine testing. 

882 

883 Recommendations for testing dried dairy products: These products are derived from milk that had been 

884 previously pasteurized. However, due to the many processing steps and transfer within and between 

885 facilities, post-process contamination can occur. Growth can occur during holding of milk or whey prior to 

886 condensation (concentration) or drying unless sufficiently temperature-time controlled. 

887 • Routine testing for standard plate counts and coliforms or Enterobacteriaceae is recommended as 

888 indicators of process control. 

889 • Due to the history of contamination, high frequency routine testing of Salmonella in RTE dried dairy 

890 products is recommended. 

891 • Testing of toxigenic microorganisms such as S. aureus or B. cereus is limited to investigative testing 

892 if hold temperature/time for fluid product before drying exceed limits identified in food safety 

893 plan. 
 

 

 

 

 

 

 

 

 

 

 

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894 • For ingredients that are used in infant formula or for other high-risk individuals, testing is more 

895 stringent and additionally includes Cronobacter and mesophilic spores or sulfite reducing 

896 Clostridium spores that may be indicators of loss of process control 

897 

898 Frozen Dairy 

899 Examples include ice cream, frozen yogurt, gelato, frozen custard. 

900 

901 Question 1. What principles and criteria should a company apply in determining the need for and in 

902 designing an effective microbial testing program to verify that processes are effectively controlling 

903 microbial pathogens? 

Criterion/Factor Frozen Dairy 

A. Are pathogens 

associated with the food 

or ingredients? 

Yes. There have been outbreaks of Salmonella and L. 

monocytogenes associated with ice cream due to environmental 

contamination or untreated ingredients (9, 27). 

B. Are the ingredients likely 

to be contaminated? 

Yes. If using ingredients like raw eggs or untreated flour in raw 

cookie dough, or untreated fruits or nuts 

C. Are there robust 

processing control 

procedures such as a kill 

step or other reduction 

methods/controls? 

Yes. Pasteurization of the ice cream mix, and treatment for some of 

the ingredients added after pasteurization, such as nuts. 
 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Frozen Dairy 

D. Is there a potential for 

recontamination from 

the handling or the 

environment? 

Yes. Pathogen presence in finished products is typically due to postpasteurization contamination from the processing environment or 

from the addition of contaminated ingredients. 

E. Does the product support 

survival or growth? 

The frozen temperature storage does not allow for growth but does 

allow for survival of pathogens such as Salmonella and L. 

monocytogenes; pathogens can grow if the product is thawed and 

held at growth temperatures for extended periods. 

F. Is this product meant for 

higher risk population? 

No, but is consumed by the elderly and immunocompromised 

G. What is the shelf life of 

the product? 

Months to years 

H.Will consumer handling 

and use increase or 

decrease likelihood of 

pathogen survival, 

growth, or toxin 

production and risk of 

consumer illness? 

In general, frozen products are intended to be consumed in the 

frozen state. However, ice cream can be used in milk shakes, which if 

left at temperatures that support growth can allow for the growth of 

L. monocytogenes. 

904 

905 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

906 enzymes, would be an appropriate verification activity? 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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907 No for most products; for frozen yogurt, monitoring acid development during the culturing process 

908 is important to inhibit growth of pathogens during fermentation. 

909 

910 Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

911 evidence that the appropriate treatment was, in fact, applied? 

912 No. This type of product has exposure to the environment, and ingredients are often added after 

913 pasteurization. 

914 

915 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

916 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

917 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

918 What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

919 Consideration should be given to the types of inclusions/additions that are added after 

920 pasteurization, and the microbial hazards associated with these. Enumeration of coliforms as an indicator 

921 organism would be appropriate (<10 or <20 CFU CFU/g for plain or frozen desserts with inclusions, 

922 respectively). Enumeration of total microbial loads (Aerobic plate count/SPC) may be variable depending 

923 on the inclusions and could range up to 4-log CFU/g and still be of acceptable quality SPC testing for frozen 

924 yogurt is impractical because the product is made with the additional of starter cultures. Due to the risk of 

925 environmental contamination, testing for Listeria spp. in the environment can be used as an indicator for 

926 L. monocytogenes. Products produced under the official USDA Quality Approved Inspection Shield must 

927 meet the requirements in 7 CFR 58.646 (no more than 50,000 CFU/g SPC, no more than 10 coliform CFU/g 

928 coliform for plain and no more than 20 coliform CFU/gram in chocolate, fruit, nut or other flavors). 

929 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

930

935

940

945

950

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Question 5. What principles and criteria should a company apply in determining the frequency of testing 

931 finished product to determine if the company’s food safety system for that product is effective? 

932 Testing frequency for frozen dairy products is dependent upon the level of control during 

933 manufacturing. Factors to consider include whether ingredients are added post-pasteurization, the design 

934 of the equipment, the condition of the facility, how much manual handling occurs, and the results of the 

environmental monitoring program. Enterobacteriaceae testing is an effective and simple tool to 

936 determine hygiene status of parts of the facility that are primarily dry, whereas Listeria spp. may be a better 

937 indicator in areas that are wet cleaned, but both microbes can be useful wherever product accumulates. 

938 

939 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

the goal of verifying the adequacy of control of microbial hazards? 

941 Samples taken at critical steps along the processing line play an important role in determining the 

942 effectiveness of preventive measures to control recontamination after the heat treatment. Samples are 

943 typically taken from the mixing and maturation tanks (tanks used to cool the pasteurized ice cream mix to 

944 4°C with mixing), at the filler or after hardening tunnels. Particular attention needs to be paid to build-up 

of residues or condensation spots where microbial growth may occur. 

946 

947 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

948 product testing verification activities by companies? 

949 A robust environmental monitoring program that demonstrates that Listeria is in control will 

reduce the need for finished product testing because they are microorganisms of concern in frozen dessert. 

951 The EMP should also periodically test for Salmonella; this pathogen is associated with dairy powders or 

952 other dry ingredients that are used in the ice cream mix prior to pasteurization and could be introduced 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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953 Salmonella into the plant environment. Verifying that this pathogen is controlled in the environment 

954 reduces the risk that it will be a post-process contaminant in the finished product. If EMP suggest that 

955 contamination of product by L. monocytogenes may have occurred, increased finished product testing is 

956 recommended; refer to government guidance documents for testing product (68). 

957 

958 Question 8: What criteria should a company apply in determining that microbial testing results indicate 

959 a loss of (systemic) process control? 

960 What actions should a company take if test results indicate a loss of control? 

961 When verification testing indicates loss of process control, to what extent should verification testing be 

962 increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

963 Because cold-filled cultured dairy products are expected to contain high populations of starter 

964 culture, testing the environment and monitoring other process controls (e.g., acid production and cooling 

965 rates), are more actionable tests of loss of process control. When acid production is slow or stalls or cooling 

966 deviations occur revealing loss of systemic process control, the company should initially investigate causes. 

967 Product should be placed on hold and evaluated to ensure there was no potential for growth of toxigenic 

968 pathogens to levels that could cause illness. Follow FDA Draft Guidance on environmental monitoring to 

969 verify control of L. monocytogenes (68) 

970 In the event of failure of environmental monitoring, corrective actions should be undertaken, and 

971 the verification testing should be increased and expanded to all areas of manufacturing to determine the 

972 source or sources of contamination. Once identified and resolved, verification testing can be reduced. 
 

 

 

 

 

 

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973 Table A-7. Microbial targets, limits, and recommended actions if limits are exceeded, for Dairy-Frozen 

974 Desserts 

Target Microorganism Microbial Limit Recommended Action 

if Limit is Exceeded 

Comments 

Coliforms <10 CFU/g for 

plain 

<20 CFU/g for 

chocolate, fruit, 

nut or other 

flavors 

§ 58.648 

Investigate, 

implement corrective 

action. 

Routine testing. 

Populations may be 

influenced by ingredients 

therefore other coliform 

levels may be appropriate. 

Aerobic plate count <50,000 for plain Investigate, Routine. Populations are 

(APC, SPC) ice cream 

§ 58.648 

implement corrective 

action. 

influenced by ingredients; 

product specific aerobic 

plate count limits need to be 

established based on 

baseline testing. It is 

impractical to use SPC for 

Frozen yogurt made with 

starter culture 
 

 

 

 

 

 

 

 

 

 

 

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Target Microorganism Microbial Limit Recommended Action 

if Limit is Exceeded 

Comments 

Listeria 

monocytogenes 

Negative in 25 g Reject lot; Investigate, 

implement corrective 

action. 

Periodic testing or 

investigative testing in 

response to elevated counts 

of indicator organisms or in 

response to environmental 

monitoring findings 

suggesting post-process 

contamination 

975 

976 Recommendations for testing frozen dairy desserts: This product category is made with pasteurized milk 

977 or cream, but contamination can be introduced during the churning/freezing process and through 

978 ingredient additions (such as fruit, nuts, chocolate, cookie dough, etc.). 

979 • Routine microbial testing for coliforms and/or aerobic plate count (SPC), but microbial limits will 

980 depend on the types of inclusions. SPC testing for frozen yogurt is not practical because it is made 

981 with starter cultures. 

982 • Pathogen testing for L. monocytogenes and Salmonella may be limited to investigative testing in 

983 response to increasing trends of indictor organisms and environmental testing results that suggest 

984 potential post-process contamination of the product 

985 • Ingredients and inclusions added after pasteurization should be obtained from approved suppliers 

986 and subjected to supplier verification activities. 

987 o Ingredients from a new supplier with little history may require addition verification testing 
 

 

 

 

 

 

 

 

 

 

990

995

1000

1005

1010

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988 Milk and Milk Products (fluid) 

989 This category represents the various types of fluid milk and milk products such as whole milk, 

reduced or low-fat milk, skim milk, and flavored milk. Milk and milk products to include all Grade “A” raw 

991 milk and/or milk products are required to be properly pasteurized, aseptically processed and packaged, or 

992 retort processed after packaging following accordance with mandatory chemical, physical, bacteriological, 

993 and temperature standards (63). During the collecting, transporting, and manufacturing processes, 

994 measures can be taken to ensure the removal, inactivation, or absence of microbial levels that would 

contribute to foodborne illness. Bacteria and toxin production are known causative agents of foodborne 

996 illness when consuming milk and milk products. In January 2019, the Centers for Disease Control and 

997 Prevention (CDC) documented the investigation of Brucella strain RB51 exposures due to consuming 

998 contaminated raw milk that spanned over 19 states with the origin determined from a farm in, 

999 Pennsylvania (10). This investigation is an example of how not exercising pasteurization measures can 

threaten the public’s health by unnecessary exposure and subsequent illness. Additionally, other 

1001 microorganisms such as Listeria and Salmonella have caused outbreaks of pasteurized dairy products in 

1002 pasteurized milk. An outbreak of Listeria monocytogenes was identified with consumption of pasteurized 

1003 milk from a Massachusetts dairy (8). Further, a Salmonella outbreak was caused from multi-drug resistant 

1004 Salmonella Typhimurium in pasteurized milk from a dairy (51). Bacteria, such as Enterobacteriaceae and 

Pseudomonas, in processed milk can result from post-pasteurization contamination (PPC)(46). PPC can 

1006 result from insufficient cleaning or sanitizing, lack of personnel good hygiene and handling practices, and 

1007 environmental issues such as standing water, milk residue, drains, and condensate (46). Because 

1008 vegetative pathogens are rarely found in properly pasteurized milk, testing for indicators organisms at 

1009 appropriate production points should be used as verification of sanitation. 
 

 

 

 

 

 

 

 

 

 

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Page A-77 of 92 

1011 

1012 

1013 

1014 

Question 1. What principles and criteria should a company apply in determining the need for and in 

designing an effective microbial testing program to verify that processes are effectively controlling 

microbial pathogens? 

Principles that apply to finished product testing of this RTE food: 

Criterion/Factor Whole milk, reduced or low-fat milk, skim milk, and flavored 

milk 

A. Are pathogens Yes, raw milk may contain multiple pathogens, including 

associated with the food Salmonella, E. coli O157:H7 and other STEC, L. 

or ingredients? monocytogenes, Campylobacter, S. aureus, Yersinia, and 

Brucella (7, 25, 55, 60) 

B. Are the ingredients Yes, 1-30% of raw bulk tank samples are positive for one or 

likely to be more pathogens including Campylobacter jejuni, shiga-toxin 

contaminated? producing Escherichia coli, Listeria monocytogenes, 

Salmonella spp., and Yersinia enterocolitica (37). 

C. Are there robust Yes, High-Temperature-Short-Time (HTST) pasteurization, 

processing control which uses a combination of time-temperature of 72oC for at 

procedures such as a kill least 15 seconds, which in the US is regulated by the PMO 

step or other reduction (63) and 21 CFR 1240.61. Ultra-High Temperature (UHT; 

methods/controls? Ultra-pasteurization), applies a high temperature (>135o

C) for 

1-2 seconds and then rapidly chilled. 

D. Is there a potential for 

recontamination from 

Low (since product exposure is minimal following 

pasteurization), if proper precautions are conducted to 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Whole milk, reduced or low-fat milk, skim milk, and flavored 

milk 

the handling or the 

environment? 

prevent transient contamination (e.g., worker contact 

without proper hygiene, exposure to biological aerosols). 

E. Does the product 

support survival or 

growth? 

Yes. Milk and milk products have optimal water activity and 

pH levels and provide nutrients to support microbial growth. 

F. Is this product meant for 

higher risk population? 

These products are primarily for the general public; however, 

high-risk populations such as children and the elderly can be 

more severely impacted if these products are contaminated. 

G. What is the shelf life of 

the product? 

HTST pasteurization process can extend the shelf-life of milk 

for up to 3 weeks, depending on the initial microbiological 

quality of the raw milk. 

Ultra-pasteurized milk and milk products have a shelf life of 

30-90 days under proper refrigeration. 

H.Will consumer handling 

and use increase or 

decrease likelihood of 

pathogen survival, 

growth, or toxin 

production and risk of 

consumer illness? 

Temperature abuse by the consumer or extended storage 

beyond use-by date could allow growth of L. monocytogenes 

if product is recontaminated after pasteurization. However, 

spoilage microbes are likely to outcompete psychrotrophic 

pathogens. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

1015

1020

1025

1030

1035

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1016 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

1017 enzymes, would be an appropriate verification activity? 

1018 Yes. The presence of non-microbiological alkaline phosphatase (ALP; an enzyme that is denatured 

1019 by milk pasteurization) in milk is an indication that pasteurization of the milk was not achieved (53, 64), 

allowing microbiological pathogens to persist, if present. However, reactivation of ALP and presence of 

1021 non-bovine milk ALP has been shown to occur, particularly in UHT milk, which leads to difficulties in using 

1022 the assay for regulatory purposes (53). The maximum level of alkaline phosphatase is limited to less than 

1023 2.0 micrograms phenol equivalents per gram (1); stated limits in the PMO is less than 350 milliunits per L 

1024 for fluid products and other milk products by approved electronic phosphatase procedures (63). 

1026 Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

1027 evidence that the appropriate treatment was, in fact, applied? 

1028 Even though products have a validated lethality step and filled in a closed environment, 

1029 microbiological verification testing is a well-established process for pasteurized milk and milk products, 

including requirements for not to exceed 20,000 SPC per ml or gram and not to excel 10 coliform per ml 

1031 (63). As prescribed under the Pasteurized Milk Ordinance (PMO), microbial risk factors are considered and 

1032 evaluated at each process step, including primary production, milk collection, storage, pasteurization, 

1033 packaging, and transportation to determine what steps are required (63). 

1034 

Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

1036 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

1037 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 
 

 

 

 

 

 

 

 

 

 

 

 

 

1038

1039

1040

1041

1042

1043

1044

1045

1046

1047

1048

1049

1050

1051

1052

1053

1054

1055

1056

1057

1058

1059

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What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

Enumeration of standard plate count (SPC, aerobic plate count) and coliform counts in milk and 

milk products verifies minimal post pasteurization bacterial contamination (63). Pathogen testing is not 

typically conducted unless results for EMP indicate risk of contamination by L. monocytogenes. 

Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

The frequency of finished product testing SPC and coliforms in Grade “A” milk and milk products is 

prescribed in the PMO (63). Per PMO (63), during any six consecutive months, a minimum of four samples 

of each product “shall be collected by the Regulatory Agency in at least four separate months, except when 

three months show a month containing two sample dates separated by 20 days.” If the production of milk 

and/or milk product is not on a continuous monthly basis, and therefore the firm cannot comply with the 

sampling requirements above, then a sample must be collected during each month of production. 

Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

the goal of verifying the adequacy of control of microbial hazards? 

None, other than an Environmental Monitoring Program (EMP). Environmental monitoring is 

critical to ensure microbial contamination is not in finished milk and milk products and is a means of 

verifying the effectiveness of the overall sanitary conditions in relation to design, method, frequency, and 

personnel practices (Innovation Center, 2019). The PMO requires a written EMP plan for milk and milk 

products exposed to environmental conditions prior to packaging; follow industry and government 

guidance for Listeria control (30, 68). 
 

 

 

 

 

 

 

 

 

 

 

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1060 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

1061 product testing verification activities by companies? 

1062 If Listeria spp. is detected in the environment, conduct investigational testing that may include 

1063 food contact surfaces and product (30, 68). Verification activities will be dependent on an evaluation of 

1064 the results, including the zone and frequency of Listeria spp. positives. 

1065 

1066 Question 8: What criteria should a company apply in determining that microbial testing results indicate 

1067 a loss of (systemic) process control? What actions should a company take if test results indicate a loss 

1068 of control? When verification testing indicates loss of process control, to what extent should verification 

1069 testing be increased, how far upstream and downstream should it go, and when and how should it be 

1070 scaled back? 

1071 If microbial limits for indicators identified by the PMO are exceeded, this could indicate loss of 

1072 process control. The PMO indicates a loss of control to be addressed in a Corrective Action Plan (CAP). 

1073 Implement the CAP, which should include increased frequency of testing the product and the 

1074 environment, as appropriate (63); investigative testing may be product testing for E. coli and pathogens, 

1075 microbial load in water used for cleaning milk contact surfaces, etc. 

1076 

1077 Table A-8. Microbial targets, limits, and recommended actions if limits are exceeded, for Milk and Milk 

1078 Products (Fluid finished product) 

Target Microorganism Microbial Limit Recommended Action if 

Limit is Exceeded 

Comments 

Aerobic plate count 

(APC, SPC) 

<2.0 x 104/ml or g Investigate, implement 

corrective action 

Routine testing per 

PMO (63) 
 

 

 

 

 

 

 

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Coliforms <10/ml Investigate, implement 

corrective action. 

if 10 or more per ml 

Routine testing per 

PMO (63) 

Listeria monocytogenes Absent in 25 g Destroy lot or divert to 

appropriate use with a 

lethality step. Investigate 

cause of contamination. 

Determine if other lots are 

involved. Determine steps to 

prevent reoccurrence. 

Investigative testing 

as response to EMP 

that suggests likely 

contamination of 

product 

1079 

1080 Recommendations for testing fluid milk products: Legal pasteurization in the US is intended to eliminate 

1081 vegetative pathogens and product exposure is minimal following pasteurization, which is likely to prevent 

1082 recontamination; risk of exposure is low. However, 

1083 • To comply with regulations, routine testing for standard plate count and coliforms is expected. 

1084 • Develop a robust environmental monitoring program for Listeria spp. as verification of sanitation 

1085 • Pathogen testing of product is not recommended unless environmental monitoring program 

1086 indicates risk of post-pasteurization contamination 
 

 

 

 

 

 

 

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insanitary conditions. J. Food Prot. 81:115-141. 

51. Olsen, S. J., M. Ying, M. F. Davis, M. Deasy, B. Holland, L. Iampietro, C. M. Baysinger, F. Sassano, L. 

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1241 Blake. 1987. Massive outbreak of antimicrobial-resistant salmonellosis traced to pasteurized milk. JAMA. 

1242 258:3269-3274. 

1243 56. Ryser, E. T., and E. H. Marth. 2007. Listeria, Listeriosis, and Food Safety, 3rd, ed. CRC Press, Taylor 

1244 and Francis Group, Boca Raton. 

1245 57. Schaack, M. M., and E. H. Marth. 1988. Behavior of Listeria monocytogenes in skim milk and in 

1246 yogurt mix during fermentation by thermophilic lactic-acid bacteria. Journal of Food Protection. 51:607-

1247 614. 

1248 58. Sonnier, J. L., J. S. Karns, J. E. Lombard, C. A. Kopral, B. J. Haley, S. W. Kim, and J. A. S. Van Kessel. 

1249 2018. Prevalence of Salmonella enterica, Listeria monocytogenes, and pathogenic Escherichia coli in bulk 

1250 tank milk and milk filters from US dairy operations in the National Animal Health Monitoring System Dairy 

1251 2014 study. J. Dairy Science. 101:1943-1956. 

1252 59. Steinbrunner, P., B. P. Marks, E. T. Ryser, Q. J. Suehr, and S. Jeong. 2021. Fate of Salmonella and 

1253 Enterococcus faecium during pilot-scale spray drying of soy protein isolate. J. Food Prot. 84:674-679. 

1254 60. Tacket, C. O., J. P. Narain, R. Sattin, J. P. Lofgren, C. Konigsberg, Jr, R. C. Rendtorff, A. Rausa, B. R. 

1255 Davis, and M. L. Cohen. 1984. A Multistate Outbreak of Infections Caused by Yersinia enterocolitica 

1256 Transmitted by Pasteurized Milk. JAMA. 251:483-486. 

1257 61. Trmcic, A., R. Ralyea, L. Meunier-Goddik, C. Donnelly, K. Glass, D. D'Amico, E. Meredith, M. 

1258 Kehler, N. Tranchina, C. McCue, and M. Wiedmann. 2017. Consensus categorization of cheese based on 

1259 water activity and pH-A rational approach to systemizing cheese diversity. Journal of Dairy Science. 

1260 100:841-847. 

1261 62. Tschape, H., R. Prager, W. Streckel, A. Fruth, E. Tietze, and G. Bohme. 1995. Verotoxinogenic 

1262 Citrobacter freundii associated with severe gastroenteritis and cases of hemolytic-uremic syndrome in a 

1263 nursery-school - green butter as the infection source. Epidemiology and Infection. 114:441-450. 
 

 

 

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63. U. S. Department of Health and Human Services. 2019. Grade “A” Pasteurized Milk Ordinance, 

Including Provisions from the Grade “A” Condensed and Dry Milk Products and Condensed and Dry 

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64. U.S. Department of Health and Human Services. 2010. Compliance Policy Guide Sec. 527.300 

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p. 50-53. In, Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins. Second Edition, 

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of Agriculture Food Safety Inspection Service. Date, 2018, Hazard Analysis and Risk-Based Preventive 

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foods: guidance for industry. Available at https://www.fda.gov/files/food/published/Draft-Guidance-forIndustry--Control-of-Listeria-monocytogenes-in-Ready-To-Eat-Foods-%28PDF%29.pdf Accessed August 

12, 2020. 

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1286 70. U.S. Food and Drug Adminstration (FDA). Date, 2017, Control of Listeria monocytogenes in 

1287 Ready-To-Eat Foods: Guidance for Industry. Available at: 

1288 https://www.fda.gov/files/food/published/Draft-Guidance-for-Industry--Control-of-Listeria1289 monocytogenes-in-Ready-To-Eat-Foods-%28PDF%29.pdf. Accessed August 12, 2020. 

1290 71. Voysey, P. A., P. A. Anslow, K. J. Bridgwater, B. Lavender, and L. Watson. 2009. The effects of 

1291 butter characteristics on the growth of Listeria monocytogenes. Int J Dairy Technol. 62:326-330. 

1292 72. Wehr, H. M., and J. F. Frank. 2004. Standard Methods for the Examination of Dairy Products, 17th 

1293 ed. In American Public Health Association, Washington, DC. 

1294 73. Weissman, J. B., R. D. Deen, M. Williams, N. Swanston, and S. Ali. 1977. An island-wide epidemic 

1295 of salmonellosis in Trinidad traced to contaminated powdered milk. West Indian Med J. 26:135-143. 

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1297 Zwietering. 2018. Factors that inhibit growth of Listeria monocytogenes in nature-ripened Gouda cheese: 

1298 A major role for undissociated lactic acid. Food Control. 84:413-418. 

1299 75. Wisconsin Cheese Makers Assocation. Date, 2020, Hygienic Zoning/Environmental Monitoring. 

1300 Available at: https://assets.noviams.com/novi-file-uploads/wcma/PDFs/SOP-14-Hygenic-Zoning1301 Environmental-Monitoring_WM.pdf. Accessed August 12, 2020, 2020. 

1302 76. Zehren, V. L., and V. F. Zehren. 1968. Relation of acid development during cheesemaking to 

1303 development of staphylococcal enterotoxin A. J. Dairy Sci. 51:645-649. 
 

 

 

 

 

 

 

 

 

 

 

 

 

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1 APPENDIX B - CATEGORY: GRAIN-BASED PRODUCTS 

2 RTE baked items, refrigerated or time-temperature controlled for safety (TCS) 

3 RTE baked items, shelf stable or non-TCS 

4 RTE cereals 

5 RTE cold-pressed bars 

6 1. RTE, baked items, refrigerated or temperature-time controlled for safety (TCS) 

7 Examples include baked or fried items that are filled after baking with cream or custard fillings such 

8 as eclairs or donuts; foods that are made from or coated with batter or have a grain-based wrapper with 

9 fillings that have high water activities (>0.94) and a neutral pH and fried or baked batter-dipped 

10 vegetables. A third type products that could be included in this category is refrigerated or frozen baked 

11 goods such as cakes, pies, muffins, brownies, waffles, pancakes, and pizza. Some of these foods are not 

12 homogeneous, and the interface between the distinct food components may allow growth of pathogens 

13 that survive cooking (e.g., spore-formers) or are contaminants from the processing environment (e.g., 

14 Listeria monocytogenes). 

15 

16 Example 1 – Refrigerated custard-filled chocolate-iced pastry (e.g., donut or éclair) 

17 Example 2 – Frozen waffles or other baked or fried foods made with batter with extended run times 

18 

19 Question 1. What principles and criteria should a company apply in determining the need for and in 

20 designing an effective microbial testing program to verify that processes are effectively controlling 

21 microbial pathogens? 

22 

23 
 

 

 

 

 

 

 

 

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24 Table B1. Criteria/principles for RTE baked items, refrigerated or TCS 

Criterion/Factor Refrigerated Custard-Filled 

Chocolate-iced Pastry 

Frozen waffles made with batter 

A. Are pathogens 

associated with the food 

or ingredients? 

Filled bakery products have been 

implicated in foodborne illnesses 

involving S. aureus, Salmonella, L. 

monocytogenes and Bacillus cereus. 

Salmonella and STEC have been 

associated with raw flour (1, 8, 9, 11, 

22). Salmonella is found in raw eggs, 

milk powders, yeast, and cocoa 

powder (12). S. aureus is a 

contaminant that may be associated 

with food handlers (19, 23). 

Ingredients used in the custard filling 

may also contain pathogenic sporeformers such as Clostridium 

botulinum, C. perfringens and B. 

cereus that must be controlled by 

refrigeration, pH, water activity 

and/or growth inhibitors (e.g., 

potassium sorbate or buffered 

vinegars). 

Salmonella, L. monocytogenes, B. 

cereus and STEC have been associated 

with raw flour. Salmonella is found in 

raw eggs. S. aureus is a contaminant 

that may be associated with food 

handlers. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Refrigerated Custard-Filled 

Chocolate-iced Pastry 

Frozen waffles made with batter 

B. Are the ingredients likely Yes. Supplier verification programs Yes. In-shell eggs are a raw 

to be contaminated? are necessary for some ingredients 

such as Salmonella in the cocoa 

powder. Each ingredient needs to be 

assessed. 

agricultural commodity and most 

flour has not been treated to 

inactivate pathogens such as 

Salmonella. Milk is pasteurized so 

there will not be vegetative 

pathogens associated with it. 

C. Are there robust Baking of the pastry shell provides Yes. The baking process will destroy 

processing control pathogen lethality. The custard filling vegetative pathogens present in the 

procedures such as a kill containing eggs will also have a batter. However, there is the 

step or other reduction lethality step. All of the ingredients in potential for S. aureus or B. cereus 

methods/controls? the chocolate icing would have 

received a lethal treatment, but icing 

ingredients are mixed with no kill step 

applied prior to adding to the baked 

pastry. Supplier controls are needed 

for the ingredients using to make the 

icing. 

enterotoxin formation in the batter 

during extended production runs (7, 

10, 13, 26). The enterotoxin will not 

be destroyed by the baking process. 

D. Is there a potential for 

recontamination from 

Yes. The product is exposed to the 

environment after baking and during 

Yes. There is the potential for L. 

monocytogenes recontamination of 
 

 

 

 

 

 

 

 

 

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Criterion/Factor Refrigerated Custard-Filled 

Chocolate-iced Pastry 

Frozen waffles made with batter 

the handling or the custard and icing production. It is the cooked waffles post-baking during 

environment? exposed to the environment during 

the chilling or freezing step prior to 

packaging. However, sanitation 

controls and a robust environmental 

monitoring program (EMP) can 

reduce the potential for the pastry to 

be contaminated with microbial 

pathogens. Of particular importance 

is preventing contamination of the 

filling after cooking/cooling with S. 

aureus from workers. Achieving a 

temperature below which S. aureus 

can grow quickly (e.g., <10°C/50°F) 

and limiting the time that the filling is 

above that temperature is important 

in preventing enterotoxin production 

(23). 

freezing and packaging. 

E. Does the product 

support survival or 

growth? 

Environmental pathogens that may 

contaminate the product would 

survive refrigeration/frozen storage. 

Vegetative pathogens such as 

Salmonella and L. monocytogenes will 
 

 

 

 

 

 

 

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NACMCF_RTETesting_Appx_B_Grains_Final_11Jul2021.docx 

Criterion/Factor Refrigerated Custard-Filled 

Chocolate-iced Pastry 

Frozen waffles made with batter 

L. monocytogenes may grow slowly in 

the high-water activity/neutral pH 

custard if the product is refrigerated 

for extended periods, but the low 

water activity of the cake and icing 

will prevent growth. Since this is a 

refrigerated or TCS product, it is 

assumed that the combination of 

water activity, pH, and/or presence of 

chemical preservatives of the custard 

filling would not be adequate prevent 

the growth of B. cereus and S. aureus 

if product were temperature abused. 

survive on frozen waffles but will not 

grow during frozen storage. 

F. Is this product meant for In most instances the product is being In most instances the product is being 

higher risk population? made for the general population but 

may be consumed by individuals in 

higher risk populations. 

made for the general population but 

may be consumed by individuals in 

higher risk populations. 

G.What is the shelf life of 

the product? 

1 week refrigerated; several months 

frozen 

18 months frozen storage 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

APPENDIX B - CATEGORY: GRAIN BASED PRODUCTS Page B-6 of 34 

NACMCF_RTETesting_Appx_B_Grains_Final_11Jul2021.docx 

Criterion/Factor Refrigerated Custard-Filled 

Chocolate-iced Pastry 

Frozen waffles made with batter 

H.Will consumer handling 

and use increase or 

decrease risk of 

pathogen survival, 

growth, or toxin 

production? 

Frozen product would be thawed 

prior to consumption and possibly 

brought to room temperature. 

Extended storage at room 

temperature may allow growth of S. 

aureus or B. cereus in the custard 

filling to levels where enterotoxin 

would be produced. 

If thawed and held refrigerated by the 

consumer for an extended time, there 

is the potential for the growth of L. 

monocytogenes that may have 

recontaminated the waffle during 

production. Heating the waffles 

would reduce L. monocytogenes that 

may have recontaminated the waffle 

during production but may not 

eliminate it. There is the potential for 

consumers to allow teething infants 

to eat frozen waffles without heating 

(e.g., toasting). 

25 

26 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

27 enzymes, would be an appropriate verification activity? 

28 There are no practical alternatives for verification testing of these products other than pathogens 

29 or indicator organisms. Control of the batter temperature or cooled cooked custard to less than 50°F 

30 would prevent enterotoxin production during extended runs (26), but temperature/time limits need to 

31 be validated under conditions mimicking production conditions. For extended runs, enumeration of S. 

32 aureus or B. cereus is preferred to enterotoxin testing, partially due to the lack of validated assays for B. 
 

 

 

 

 

 

 

 

 

 

 

 

35

40

45

50

55

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33 cereus enterotoxins, or the specialized equipment needed for some assays (24), such as cereulide (emetic 

34 toxin by Bacillus cereus). 

36 Question 3. Are there situations where [microbial] verification testing would not be necessary if there 

37 is evidence that the appropriate treatment was, in fact, applied? 

38 No. Although temperature/times of baking and cooking that are needed for quality exceed those 

39 required for lethality of pathogens, these products (baked pastry with custard filling and icing and cooked 

waffle) are exposed to the environment after lethality treatment, including during filling with cooled, 

41 cooked custard and adding icing after baking. 

42 Even though the outside surfaces of these products typically have water activity values that are 

43 lower than values that support rapid growth of pathogens, the custard filling is not likely formulated to 

44 prevent pathogen outgrowth (L. monocytogenes during refrigeration or S. aureus if temperature abused). 

Therefore, testing for indicator organisms of in-process (such as batter or cooled custard) should be 

46 conducted in addition to the environmental monitoring and supplier control programs. 

47 

48 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

49 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

51 What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

52 The temperature and time limits for extended runs should be based on validation studies to 

53 ensure <3-log growth of toxigenic microorganisms such as S. aureus or B. cereus (16, 26). Verification 

54 testing should include enumeration of S. aureus of the custard filling either in the finished product or from 

work in process (WIP) as appropriate, although the short shelf life of this refrigerated product may 

56 preclude having test results before shipping the product. Enumeration of S. aureus and/or B. cereus in the 
 

 

 

 

 

 

 

 

 

 

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

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raw waffle batter during extended production runs will provide an indication of the risk of enterotoxin 

production during that run and should be <103 CFU/g (15). There is the risk of product buildup in the 

kettles/tanks that may be contaminated with S. aureus or B. cereus that is not removed by the routine 

flow of fresh product in the line during extended runs. If visible, these areas could be sampled for S. aureus 

and/or B. cereus. 

Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

Ability to meet temperature/time limits to prevent growth of toxigenic microbes during extended 

runs (e.g., S. aureus, B. cereus) and lethality of infectious microorganisms during baking (e.g., Salmonella), 

along with environmental monitoring program results, and supplier control for ingredients that are added 

after lethality, should be used to determine type and frequency of finished product testing. 

Enumeration testing of the raw batter for S. aureus or B. cereus may be appropriate, depending 

on the rigorousness of the validation testing and temperature controls of the batter during production. 

Products that have a validated lethality step do not need routine microbial testing for finished product. 

Investigative testing is needed when environmental monitoring for Listeria spp. or Salmonella suggests 

insufficient sanitation or inadequate supplier control for incoming ingredients used as post-lethality 

additions (such as cold blended icing or other toppings). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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76 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

77 the goal of verifying the adequacy of control of microbial hazards? 

78 Testing of the custard filling for aerobic plate counts at or prior to the filling point into the pastry 

79 may be more appropriate than enumerating S. aureus and/or B. cereus in the finished product as a 

80 verification that no post-cook contamination or microbial growth occurred (spores may survive cooking). 

81 Enumeration of S. aureus and/or B. cereus in the raw waffle batter is a verification that microbes 

82 that can produce heat-stable enterotoxin did not grow in the batter as part of an investigation of loss of 

83 process control. Aerobic plate counts are not appropriate for waffle batter due to the initial high 

84 background flora in the batter. 

85 

86 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

87 product testing verification activities by companies? 

88 RTE baked products have a microbial reduction step (baking, cooking) prior to packaging but 

89 recontamination of the final product is possible from the environment. Therefore, a robust environmental 

90 monitoring program that demonstrates that Listeria and Salmonella are in control will reduce the need 

91 for finished product testing. 

92 If results from an environmental monitoring program suggest potential for contamination of the 

93 finished product, it could result in the increased need for microbiological testing of product as part of 

94 investigative testing or root cause analysis (25, 27). 

95 

96 Question 8: What criteria should a company apply in determining that microbial testing results indicate 

97 a loss of (systemic) process control? 

98 What actions should a company take if test results indicate a loss of control? 
 

 

 

 

 

 

 

 

 

 

 

 

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99 When verification testing indicates loss of process control, to what extent should verification testing be 

100 increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

101 If S. aureus and/or B. cereus numbers exceed a set limit in the pastry filling or in the waffle batter, 

102 investigation into the cause is warranted. Corrective actions would need to be taken for S. aureus and/or 

103 B. cereus contamination levels exceeding a set limit (see Tables B2 and B3). Corrective actions should be 

104 implemented for detection of Listeria in the environment; corrective actions followed by repeat positives 

105 may indicate the need for product testing for L. monocytogenes (see FDA draft guidance on Control of 

106 Listeria monocytogenes in RTE Foods)(27). 

107 

108 Table B2. Example of product testing for the custard in a filled pastry 

Target 

Microorganism 

Microbial Limit Recommended Action 

if limit is exceeded Comments 

Aerobic plate 

count 

<100 CFU/g Investigate reason for 

exceeding limit and 

implement corrective 

action. 

Routine testing. Populations 

may include sporeforming 

bacteria that can survive 

cooking. 

S. aureus <104 CFU/g Destroy lot. 

Investigate cause of 

contamination. 

Determine if other 

lots involved. 

Determine steps to 

prevent recurrence. 

Non-routine testing. Test as part 

of investigative action if loss of 

process control (timetemperature control during 

production) is suspected. 

B. cereus <104 CFU/g 
 

 

 

 

 

 

 

 

 

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109 Table B3. Example of product testing for batter 

Target 

Microorganism 

Microbial Limit Recommended Action if 

limit is exceeded Comments 

S. aureus <104 CFU/g Destroy lot. Investigate 

cause of contamination. 

Determine if other lots 

involved. Determine 

steps to prevent 

recurrence. 

Non-routine testing. Test as 

part of investigative action if 

loss of process control is 

suspected, such as exceeding 

temperature/time limits 

identified by challenge study. 

For extended runs, if 

temperature exceeds 

10°C/50°F, routine testing for 

S. aureus and B cereus is 

recommended. 

B. cereus <104 CFU/g 

110 

111 Recommendations for RTE, baked items, refrigerated or temperature-time controlled for safety (TCS) 

112 1. Because there is a kill step for the both the refrigerated filled pastries and the frozen waffle, no 

113 finished product testing is needed when an effective EMP program is in place. Finished product 

114 testing may be implemented when EMP results show potential for contamination of the finished 

115 product. 

116 2. Waffle batter temperature should be kept below 10°C/50°F to prevent outgrowth of S. aureus 

117 and/or B. cereus such that toxin production is prevented. For extended runs where batter 
 

 

 

 

 

 

 

 

 

 

120

125

130

135

140

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118 temperature is greater than 10°C/50°F, routine enumeration testing of the batter for S. aureus 

119 and/or B. cereus is recommended based on results from validation studies for extended runs. 

3. For RTE baked goods where components do not have a microbial reduction step (such as a cold121 blended icing or toppings), ingredients should be obtained from approved suppliers and subjected 

122 to supplier verification activities. Ingredients from a new supplier with little history may require 

123 additional verification testing. 

124 

2. RTE, baked items, shelf stable or non-TCS 

126 Examples include fully baked manufactured from dough or batter such as bread (flat and 

127 leavened), cookies, crackers, pretzels, wafer or waffle cones, and certain pastries, cakes, and pies with 

128 validation studies showing they are exempt from TCS requirements. Some may have inclusions that are 

129 baked with the item such as fruits, vegetables, or cheeses or are iced or filled with shelf stable (low water 

activity) components such as jellies after baking. There may be leavening of the dough or batter, either 

131 through yeast fermentation or chemical leavening agents. Stability of these types of products is achieved 

132 by one of or combinations of lowered pH, reduced water activity or chemical agents to prevent mold 

133 growth. 

134 

Example 1 – Chocolate, creme-filled sandwich cookie 

136 Example 2 – Whole wheat, sliced bread 

137 

138 Question 1. What principles and criteria should a company apply in determining the need for and in 

139 designing an effective microbial testing program to verify that processes are effectively controlling 

microbial pathogens? 
 

 

 

 

 

 

 

 

 

 

 

 

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141 Table B4. Criteria/principles for RTE, baked items, shelf stable or non-TCS 

Criterion/Factor Chocolate, creme-filled sandwich 

cookie 

Whole wheat, sliced bread 

A. Are pathogens Salmonella, L. Salmonella, L. 

associated with the monocytogenes, STEC, and monocytogenes, STEC, and 

food or ingredients? pathogenic spore-formers have 

been associated with raw flour (1, 

22); Salmonella has been found in 

cocoa powder (6). 

pathogenic spore-formers have 

been associated with raw flour and 

seeds (28) 

B. Are the ingredients Yes. The flour is likely to be Yes. Most flour is not treated and is 

likely to be contaminated with spore-formers, likely to be contaminated with 

contaminated? but the incidence is generally low 

for vegetative pathogens. Cocoa 

powder could be contaminated if 

supplier has not applied proper 

process controls and 

environmental control programs to 

prevent recontamination. Flour and 

cocoa powder will receive a kill 

step (baking cookie). Processed 

ingredients used in the crèmefilling (sugar, oils) have low 

likelihood of contamination. 

vegetative pathogens and 

pathogenic spore-formers. 

Salmonella and L. monocytogenes 

have been associated with seeds 

that may be used as bread toppings 

(applied prebake). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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C. Are there robust 

processing control 

procedures such as a 

kill step or other 

reduction 

methods/controls? 

Baking of the cookie provides 

pathogen lethality, but no kill step 

is applied to the finished product 

after adding filling and assembly. 

Yes. Baking of bread will provide 

greater than 5-log destruction of 

vegetative pathogens (3, 21). 

D. Is there a potential for Yes. The product is exposed to the Yes. The product is exposed to the 

recontamination from environment after baking and environment after baking and prior 

the handling or the during icing/filling and prior to to packaging. However, sanitation 

environment? packaging. However, sanitation 

controls and a robust EMP can 

reduce the potential for the 

sandwich cookie to be 

contaminated with pathogens. 

controls and a robust EMP can 

reduce the potential for the bread 

to be contaminated with microbial 

pathogens. 

E. Does the product 

support survival or 

growth? 

Environmental pathogens that may 

contaminate the product would 

survive storage. Growth of 

pathogens would not be possible 

due to the low water activity of the 

cookie and filling. Spore-formers 

can survive, but not grow. 

Environmental pathogens that may 

contaminate the product exterior 

would survive storage. Growth of 

pathogens would not be possible 

due to the low water activity of the 

exterior crust. Spore-formers can 

survive, but not grow. 
 

 

 

 

 

 

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F. Is this product meant 

for higher risk 

population? 

In most instances the product is 

being made for the general 

population but may be consumed 

by individuals in higher risk 

populations. 

In most instances the product is 

being made for the general 

population but may be consumed 

by individuals in higher risk 

populations. 

G. What is the shelf life of 

the product? 

9-12 months 1-4 weeks depending on use of 

preservatives 

H. Will consumer handling 

and use increase or 

decrease risk of 

pathogen survival, 

growth, or toxin 

production? 

Consumer use is not likely to affect 

the risk of pathogens on this 

product. 

Consumer use is not likely to affect 

the risk of pathogens on this 

product. 

142 

143 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

144 enzymes, would be an appropriate verification activity? 

145 No other testing is appropriate beyond verification that temperature/time limits for lethality have 

146 been met. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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147 Question 3. Are there situations where [microbial] verification testing would not be necessary if there 

148 is evidence that the appropriate treatment was, in fact, applied? 

149 Although these products are exposed to the environment after a validated lethality process 

150 (baking), the products have low water activity that will not support the growth of pathogens from 

151 recontamination after the baking kill step and filling of the sandwich cookie throughout their shelf-life. 

152 Neither product has been associated with foodborne outbreaks. Microbial testing of finished product is 

153 not needed when an effective EMP program is in place and ingredients added after baking are obtained 

154 from approved suppliers and subjected to supplier verification activities. 

155 

156 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

157 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

158 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

159 What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

160 Routine finished product testing is not needed for shelf-stable cookies. Mold counts for breads 

161 can be helpful as a quality indicator, but frequently retaining loaves for incubation and visual inspections 

162 for mold growth within shelf-life is used as an alternative to enumeration. 

163 

164 Question 5. What principles and criteria should a company apply in determining the frequency of testing 

165 finished product to determine if the company’s food safety system for that product is effective? 

166 Ability to meet temperature/time limits for lethality of infectious microorganisms during baking 

167 (e.g., Salmonella), along with environmental monitoring program results, and supplier control for 

168 ingredients that are added after lethality, should be used to determine whether to conduct finished 

169 product testing. Results from the EMP for Enterobacteriaceae or Salmonella that demonstrate sanitary 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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170 control of the processing environment preclude testing of finished product. Air sampling/monitoring of 

171 yeast and mold levels within the plant environment will help to gauge potential impact to spoilage. 

172 

173 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

174 the goal of verifying the adequacy of control of microbial hazards? 

175 No, if testing occurs, target should be after the microbial reduction step (baking). 

176 

177 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

178 product testing verification activities by companies? 

179 RTE baked products have a microbial reduction step prior to packaging but recontamination of 

180 the final product is possible from the environment. Salmonella can be introduced into the plant 

181 environment from flour and become established in the facility. Therefore, a robust environmental 

182 monitoring program that demonstrates that Salmonella (or Enterobacteriaceae as an indicator of hygiene) 

183 are in control will reduce the need for finished product testing. 

184 If results from an environmental monitoring program suggests potential for contamination of the 

185 finished product, it could result in the increased need for microbiological testing of product as part of 

186 investigative testing or root cause analysis (4, 5, 11, 20, 25). 

187 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

190

195

200

205

210

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188 Question 8: What criteria should a company apply in determining that microbial testing results indicate 

189 a loss of (systemic) process control? 

What actions should a company take if test results indicate a loss of control? 

191 When verification testing indicates loss of process control, to what extent should verification testing be 

192 increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

193 Microbiological testing of this finished product is not recommended. Monitoring of baking times 

194 and temperatures is adequate to find loss of process control. Measurement of finished product moisture 

can be evidence that an adequate baking process was applied due to the expected moisture loss during 

196 baking. EMP results (e.g., repeat positive Enterobacteriaceae or Salmonella spp.) could indicate a loss of 

197 sanitation control and could lead to investigative product testing. 

198 

199 Recommendations for RTE, baked items, shelf stable or non-TCS: Because there is a kill step for both the 

cookie and bread, and because the crème filling in the cookie is not likely to contain pathogens, finished 

201 product testing for pathogens is not needed when a robust EMP is in place. Finished product testing may 

202 be implemented when EMP results show repeat positive Enterobacteriaceae or Salmonella species. 

203 1. Because there is a kill step for RTE baked goods, no finished product testing is needed when the 

204 baking step is under control and an effective EMP program is in place. Finished product testing 

may be implemented when EMP results show potential for contamination of the finished product. 

206 2. For RTE baked goods where components do not have a microbial reduction step (such as a cold207 blended icing), ingredients should be obtained from approved suppliers and subjected to supplier 

208 verification activities. Ingredients from a supplier with little history may require additional 

209 verification testing. 

211 
 

 

 

 

 

 

 

 

 

 

 

 

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212 3. RTE Cereals 

213 Examples include breakfast cereals with or without inclusions such as nuts, and/or dried fruits, 

214 infant cereal, oatmeal, and rice cakes. This product has a cook step for some of the ingredients (e.g., 

215 grains, nuts, other inclusions) that eliminates pathogens of concern in the ingredients, but also may have 

216 added ingredients that have not received a kill step, e.g., dried fruit. The product is also exposed during 

217 preparation and filling of containers and could be contaminated with Salmonella (2, 12, 18) or L. 

218 monocytogenes. 

219 Example 1 – rice-based cereal with processed nut inclusion and dried fruit inclusion with no kill step 

220 Example 2 - infant cereal 

221 

222 Question 1. What principles and criteria should a company apply in determining the need for and in 

223 designing an effective microbial testing program to verify that processes are effectively controlling 

224 microbial pathogens? 

225 Table B5. Criteria/principles for cereals 

Criterion/Factor Rice-based cereal with processed 

nut inclusion and dried fruit 

inclusion with no kill step 

Dry infant cereal 

A. Are pathogens 

associated with the 

food or ingredients? 

Salmonella, L. monocytogenes, B. 

cereus, and STEC have been 

associated with grains. 

Salmonella, STEC, and viral 

pathogens may be associated 

with nuts or dried fruit. 

Salmonella has been associated 

with dry infant cereal (17). 

Other organisms such as S. 

aureus, B. cereus, or 

Cronobacter spp. may be 

present at low levels (29). 
 

 

 

 

 

 

 

 

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Criterion/Factor Rice-based cereal with processed 

nut inclusion and dried fruit 

inclusion with no kill step 

Dry infant cereal 

B. Are the ingredients 

likely to be 

contaminated? 

Yes Yes 

C. Are there robust 

processing control 

procedures such as a 

kill step or other 

reduction 

methods/controls? 

Yes. The grains have a lethality 

step during processing. The nuts 

are treated by the supplier. 

However, no lethality step for 

dried fruit added post-lethality 

step. 

Yes. The grains have a lethality 

step during processing. 

D. Is there a potential for 

recontamination from 

the handling or the 

environment? 

Yes. The product may be exposed 

to the environment after the 

lethality processing step prior to 

packaging. 

Yes. The product may be 

exposed to the environment 

after the lethality processing 

step prior to packaging. 

E. Does the product 

support survival or 

growth? 

Yes. Pathogens such as 

Salmonella will survive but will 

not grow. 

Yes. Pathogens such as 

Salmonella will survive but will 

not grow. 

F. Is this product meant 

for higher risk 

population? 

This product is made for the 

general population. However, 

high-risk populations may 

purchase cereal or be served 

Yes. This product is meant for 

infants aged 4 months and 

higher. 
 

 

 

 

 

 

 

 

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Criterion/Factor Rice-based cereal with processed 

nut inclusion and dried fruit 

inclusion with no kill step 

Dry infant cereal 

cereal in hospital or nursing 

home facilities. 

G. What is the shelf life 

of the product? 

18 months 18 months 

H. Will consumer 

handling and use 

increase or decrease 

risk of pathogen 

survival, growth, or 

toxin production? 

Consumer handling is unlikely to 

alter pathogen survival or 

growth. 

Consumer handling may alter 

pathogen survival or growth if 

the cereal is held for extended 

time and not temperaturecontrolled after reconstitution. 

226 

227 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

228 enzymes, would be an appropriate verification activity? 

229 No 

230 

231 Question 3. Are there situations where [microbial] verification testing would not be necessary if there 

232 is evidence that the appropriate treatment was, in fact, applied? 

233 No, finished product testing is required for the cereals (except the infant cereal) if supplier 

234 verification supports the adequacy of supplier controls for the inclusions and an appropriate 

235 environmental monitoring program shows that the process is under control. Dried cereals are low water 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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236 activity products that do not support the growth of pathogens. However, dried cereals could allow 

237 persistence of pathogens such as Salmonella (if present). 

238 For the infant cereal, when environmental testing results are negative for Salmonella, testing of 

239 end product for Salmonella is still appropriate, Table B7 (12). 

240 

241 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

242 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

243 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? What are 

244 appropriate indicator microorganisms for verifying processes adequately control pathogens? 

245 When finished product testing is done (depending on EMP results or if product is intended for 

246 high-risk individuals), presence/absence of Salmonella would be the appropriate organism. Product 

247 should be held until testing is complete. 

248 

249 Question 5. What principles and criteria should a company apply in determining the frequency of testing 

250 finished product to determine if the company’s food safety system for that product is effective? 

251 Routine testing is not needed for shelf-stable cereals not intended for high-risk individuals. 

252 However, if environmental testing indicates the presence of Salmonella, then finished product should be 

253 tested. In addition, testing should occur in zones 1 and 2, including vitamin or sugar spray nozzles, if used. 

254 For infant cereal, end product testing is appropriate because infants are a higher risk population and 

255 because of the risk for consumer mishandling, see Table B7. 

256 

257 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

258 the goal of verifying the adequacy of control of microbial hazards? 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

260

265

270

275

280

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259 Environmental monitoring is needed to demonstrate control of the environment. For postlethality step added ingredients, COAs should be received from suppliers and supplier control programs 

261 verified. 

262 

263 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

264 product testing verification activities by companies? 

Environmental monitoring takes place primarily in zones 2 and 3 (and in zone 1 if zone 2 is 

266 contaminated). If there is a positive for an indicator (Enterobacteriaceae) and/or Salmonella in zone 2 or 

267 3, then additional swabbing using 3D-vectoring should be conducted to look for niches. The production 

268 environment should be cleaned and sanitized, followed by subsequent testing to evaluate cleaning 

269 procedures and whether equipment needs to be altered to prevent niches (e.g., hollow rollers). If 

subsequent zone 2 sampling is positive, finished product testing may be warranted. If finished product is 

271 tested, the entire line would be cleaned before and after testing and the lot should be held until results 

272 confirm as negative. 

273 

274 Question 8: What criteria should a company apply in determining that microbial testing results indicate 

a loss of (systemic) process control? What actions should a company take if test results indicate a loss 

276 of control? When verification testing indicates loss of process control, to what extent should 

277 verification testing be increased, how far upstream and downstream should it go, and when and how 

278 should it be scaled back? 

279 If Salmonella is found in zone 2, then additional testing (e.g., every 3 or 4 days, or more frequently 

depending on findings and risks) and cleaning of zone 2 in the area where the positive was found, as well 

281 as nearby areas in zone 3, is warranted until consecutive zone 2 samples are negative. Consider testing 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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282 zone 1 and finished product. If finished product testing of infant cereal indicates that Salmonella is 

283 present, the lots must be destroyed, and an investigation into the cause of the contamination must occur. 

284 

285 Recommendations for RTE Cereals: 

286 1. Microbiological testing of certain ingredients (i.e., those that could potentially be contaminated 

287 with pathogens), the environment, and, to a limited extent, finished product, should play a role 

288 in the verification of control measures for cereal. 

289 2. Ingredients added after lethality step should be obtained from approved suppliers and subjected 

290 to supplier verification activities, which may include pathogen testing. Ingredients from a new 

291 supplier with little history may require additional verification testing. 

292 3. A robust Salmonella environmental monitoring program for all cereal products is recommended. 

293 4. Routine finished product testing for Salmonella should be conducted for dry infant cereal. 

294 Although there is a kill step for the dry infant cereal, finished product testing is warranted because 

295 the ultimate consumer, infants, is a high-risk consumer category. 

296 

297 

298 Table B6. Example of end product testing for cereal if there is suspected loss of process control 

Target 

Microorganism 

Microbial Limit Recommended Action if Limit is 

Exceeded Comments 

Salmonella Negative in 10 samples 

(case 11 sampling plan) 

(12) 

Destroy lot, investigate cause of 

contamination, determine if 

other lots involved, determine 

steps to prevent reoccurrence. 

Sample size is 25 g 

for Salmonella. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

299 

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Table B7. Example of end product testing for infant cereal 

Target 

Microorganism 

Microbial Limit Recommended Action if Limit is 

Exceeded Comments 

Salmonella Negative in 60 

samples (12) 

Destroy lot, investigate cause of 

contamination, determine if other 

lots involved, determine steps to 

prevent reoccurrence. 

Sample size is 25 g for 

Salmonella. Typically, 

four 375 g composites 

are tested for 

Salmonella. 

300 

301 4. Grain-based products: RTE, cold-pressed bars. 

302 Examples include granola bars. Cold-pressed bars are made from cooked grains, carbohydrate303 based binders, and inclusions such as fruit, nuts, and/or chocolate. The ingredients should be verified for 

304 microbiological safety, since in most cases the bars will not receive a validated lethality step during 

305 manufacturing. These added ingredients should come with a COA that includes pathogen testing. The 

306 preventive control supplier program also requires an annual onsite audit when the supplier controls a 

307 hazard that could cause serious adverse health consequences or death. Recommendations for finished 

308 product and environmental testing by suppliers depend on the specific ingredient being supplied. 

309 

310 Question 1. What principles and criteria should a company apply in determining the need for and in 

311 designing an effective microbial testing program to verify that processes are effectively controlling 

312 microbial pathogens? 

313 

314 
 

 

 

 

 

 

 

 

 

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315 Table B8. Criteria/principles for cereal bars 

Criterion/Factor Example: cold-pressed bar with chocolate and coconut 

A. Are pathogens associated 

with the food or 

ingredients? 

Salmonella has been associated with cereal, coconut, nuts, and 

chocolate (14, 20). 

B. Are the ingredients likely 

to be contaminated? 

Yes, if the supplier does not process ingredients to eliminate 

pathogens from the product or prevent recontamination. 

C. Are there robust 

processing control 

procedures such as a kill 

step or other reduction 

methods/controls? 

No, for the finished product. Many of the ingredients may have 

received a lethality treatment, e.g., chocolate, treated nuts, 

cooked grains. 

D. Is there a potential for 

recontamination from 

the handling or the 

environment? 

Yes 

E. Does the product support 

survival or growth? 

Pathogens will survive, but not grow, on dry product. 

F. Is this product meant for 

higher risk population? 

This product is made for the general population. However, highrisk populations may purchase or be served this product. 

G. What is the shelf life of 

the product? 

18 months 
 

 

 

 

 

 

 

 

 

 

 

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H. Will consumer handling 

and use increase or 

decrease risk of 

pathogen survival, 

growth, or toxin 

production? 

Consumer handling is unlikely to alter pathogen survival, growth, 

or toxin production. 

316 

317 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

318 enzymes, would be an appropriate verification activity? 

319 There are no alternatives to microbial testing per se. Robust environment monitoring should be 

320 implemented. Ingredients are tested for pathogens by supplier, CoA is provided, and supply chain controls 

321 should be verified. 

322 

323 Question 3. Are there situations where [microbial] verification testing would not be necessary if there 

324 is evidence that the appropriate treatment was, in fact, applied? 

325 Cereal bars are low water activity products that do not have a lethality treatment, although many 

326 of the ingredients will have previously been treated. But, since cereal is a low water activity food, 

327 pathogens such as Salmonella (if present) will persist. Exclusion of pathogens from the ingredients and 

328 the environment should be verified through supplier controls and environmental controls (i.e., sanitation 

329 controls verified with an EMP). 

330 

331 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

332 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

335

340

345

350

355

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333 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? What are 

334 appropriate indicator microorganisms for verifying processes adequately control pathogens? 

Enterobacteriaceae enumeration (e.g., <100/g) can be used as an indicator of potential 

336 contamination. When non-routine finished product testing for pathogens is done (e.g., quarterly), 

337 Salmonella (presence/absence in 375-g analytical unit composed of 15 x 25-g samples) would be the 

338 appropriate organism. Product would be held until testing is done. 

339 

Question 5. What principles and criteria should a company apply in determining the frequency of testing 

341 finished product to determine if the company’s food safety system for that product is effective? 

342 The frequency of finished product testing would depend, in part, on the history of environmental 

343 testing. Consistently low counts of Enterobacteriaceae and infrequent findings of Salmonella in the 

344 environment support less frequent finished product testing. If environmental testing (e.g., zones 2 and 3) 

indicates the presence of Salmonella, then investigation and testing of zone 1 and finished product should 

346 be considered. 

347 

348 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

349 the goal of verifying the adequacy of control of microbial hazards? 

Since contamination of cereal bars is from the ingredients or the environment, testing should 

351 focus on the ingredients and the environment rather than finished product. 

352 

353 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

354 product testing verification activities by companies? 

Environmental monitoring is needed. Low level environmental positives for Enterobacteriaceae 

356 or other indicator organisms do not result in the need for finished product testing, but may indicate a 
 

 

 

 

 

 

 

 

 

 

 

 

 

360

365

370

375

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357 need for increased environmental monitoring (11). If an appropriate environmental monitoring plan is 

358 implemented, no routine testing of finished product is needed. With cold-pressed cereal bars, safety is 

359 primarily addressed through supplier controls for ingredients and controls related to the environment, 

such as sanitation controls and sanitary operations, verified with environmental monitoring. If 

361 environmental monitoring indicates that the process environment is not adequate, further testing is 

362 needed to identify the point(s) in the process that need correction. The results may indicate a need for 

363 product testing for pathogens such as Salmonella. 

364 

Question 8: What criteria should a company apply in determining that microbial testing results indicate 

366 a loss of (systemic) process control? What actions should a company take if test results indicate a loss 

367 of control? When verification testing indicates loss of process control, to what extent should 

368 verification testing be increased, how far upstream and downstream should it go, and when and how 

369 should it be scaled back? 

If Salmonella is found in zone 2 or 3, then additional testing (e.g., every 3 or 4 days or more 

371 frequently) and cleaning of zone 2 or 3 is warranted until consecutive samples are negative. Consider 

372 testing zone 1 and finished product. 

373 

374 Recommendations: 

Based on the above, we recommend that: 

376 1. Microbiological testing of ingredients, the environment, and, to a limited extent, non-routine 

377 testing of finished product, should play a role in the verification of control measures for cold378 pressed bars. 

379 2. Cold-pressed bar manufacturers should conduct activities to verify that suppliers have 

implemented control measures to minimize the potential for pathogens to be present in those 
 

 

 

 

 

 

 

 

 

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381 ingredients for which pathogens have been associated. Supplier verification activities could 

382 include microbiological testing of ingredients; the frequency of such testing should be based on 

383 an assessment of the likelihood of the ingredients supplied being contaminated, considering the 

384 likelihood of contamination of the raw material for the ingredients supplied and the control 

385 programs implemented by the supplier. 

386 3. Robust environmental monitoring programs should be implemented to ensure that the cold387 pressed bars are not contaminated from the processing environment. Having a robust 

388 environmental monitoring program minimizes the need for finished product testing for 

389 pathogens. 

390 4. Routine finished product testing for pathogens is not recommended. Microbiological testing of 

391 finished product for Salmonella should be conducted if there is suspected loss of environmental 

392 or process control to investigate process and sanitation control. 

393 

394 Table B9. Example of product testing for cold-pressed bars if there is suspected loss of process control 

Target Microorganism Microbial Limit Recommended Action 

if Limit is Exceeded Comments 

Salmonella Negative in 10 samples 

(see case 11 sampling 

plan) (12) 

Destroy lot, investigate 

cause of 

contamination, 

determine if other lots 

involved, determine 

steps to prevent 

reoccurrence. 

Sample size is 25 g for 

Salmonella. 
 

 

 

 

 

 

395

400

405

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399 Agona infections linked to toasted oats cereal - United States, April-May, 1998 (Reprinted from MMWR, 

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402 Evaluation of thermal inactivation parameters of Salmonella in whole wheat multigrain bread. Food 

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409 validation and environmental monitoring. Food Prot Trends. 29:493-508. 

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416 thermal death kinetics of enterohemorrhagic Escherichia coli Serogroups O26, O103, O111, and O157 in 

417 wheat flour. Appl Environ Microbiol. 84:11. 
 

 

 

 

 

 

 

 

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New York. 

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15. National Advisory Committee for Microbiological Criteria for Foods. 2018. Response to 

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442 unsweetened cereal consumption, United States, 2008. J. Food Prot. 76:227-230. 

443 19. Schelin, J., N. Wallin-Carlquist, M. T. Cohn, R. Lindqvist, G. C. Barker, and P. Radstrom. 2011. The 

444 formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment. 

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447 Post, L. Smoot, S. Hood, J. Shebuski, and J. Banks. 2009. Control of Salmonella in low-moisture foods I: 

448 Minimizing entry of Salmonella into a processing facility. Food Prot Trends. 29:342-353. 

449 21. Shrestha, S., W. Birbari, T. Sheehan, and K. Glass. 2016. Thermal inactivation of salmonella on 

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451 22. Sperber, W. H., and North American Millers' Association Microbiology Working Group. 2007. 

452 Role of microbiological guidelines in the production and commercial use of milled cereal grains: a 

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454 23. Stewart, C. M., M. B. Cole, and D. W. Schaffner. 2003. Managing the risk of staphylococcal food 

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456 24. Tallent, S. M., A. Knolhoff, E. J. Rhodehamel, S. M. Harmon, N. Belay, D. B. Shah, and R. W. 

457 Bennett. 2020. BAM Chapter 14: Bacillus cereus. In, Bacteriological Analytical Manual (BAM) U.S. Food 

458 and Drug Adminstration,, https://www.fda.gov/food/laboratory-methods-food/bam-chapter-14-

459 bacillus-cereus. 

25. U.S. Food and Drug Administration. 2015. Investigations Operations Manual 2015, Ch. 4 

461 Sampling.http://www.fda.gov/downloads/ICECI/Inspections/IOM/UCM123507.pdf. Accessed June 13, 

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463 26. U.S. Food and Drug Administration. 2020. Chapter 15: Staphylococcus aureus Toxin Formation in 

464 Hydrated Batter Mixes. In, Fish and Fishery Products Hazards and Controls, Fourth Edition, 

465 https://www.fda.gov/media/80319/download. 

466 27. U.S. Food and Drug Adminstration. 2017. Control of Listeria monocytogenes in ready-to-eat 

467 foods: guidance for industry. Available at https://www.fda.gov/files/food/published/Draft-Guidance-for468 Industry--Control-of-Listeria-monocytogenes-in-Ready-To-Eat-Foods-%28PDF%29.pdf Accessed August 

469 12, 2020. 

470 28. Van Doren, J. M., D. Kleinmeier, T. S. Hammack, and A. Westerman. 2013. Prevalence, serotype 

471 diversity, and antimicrobial resistance of Salmonella in imported shipments of spice offered for entry to 

472 the United States, FY2007–FY2009. Food Microbiol. 34:239-251. 

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474 sakazakii (Enterobacter sakazakii) presence in cereal infant foods. Progress in Nutrition. 22:596-602. 
 

 

 

 

 

 

 

 

 

 

 

 

 

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APPENDIX C - CATEGORY: READY-TO-EAT MEALS 

This category includes a wide range of multi-component refrigerated or frozen food products, 

with shelf lives ranging from less than one week to several months. Some of these may be “heat and eat” 

foods but are still considered RTE; the food has been processed to control pathogens but is intended to 

be heated for palatability. The microbial diversity and populations depend on the ingredients used and 

handling prior to packaging, which can introduce contamination. In most cases, microbial testing uses 

hygiene indicator organisms (e.g., coliforms, Enterobacteriaceae, generic E. coli), with defined limits 

outlined in this document and in other references (1, 2, 8, 9, 12). 

RTE foods having no terminal lethality step, and with short shelf life, will rely more heavily on 

supplier controls and environmental controls, primarily verified using indicator organisms, because of the 

impracticality of holding samples until pathogen testing can be complete. In contrast, meals with longer 

shelf life that have been previously associated with Salmonella or L. monocytogenes may incorporate 

testing for pathogens as a verification of process control, as well as testing for indicator organisms. 

Environmental testing for Listeria spp. as an indicator for L. monocytogenes is common for these foods. If 

Listeria spp. is found in Zone 1 environmental samples, investigational testing for L. monocytogenes may 

be indicated (20). 

A. RTE Deli salads 

Examples include macaroni salad, potato salad, egg salad, coleslaw, 3-bean salad, and grainsbased salads (e.g., quinoa, barley). Most have a low-acid component that has been cooked (using a heat 

treatment that provides lethality for microbial pathogens), a vinegar- or mayonnaise-based dressing that 

reduces the pH but may not be sufficient to prevent growth of Listeria monocytogenes (if pH >4.4) or 

Salmonella (if pH>3.7). Most also contain added ingredients that may not have received a kill step for 

pathogens such as L. monocytogenes and Salmonella, e.g., cut vegetables such as onions, celery, and 
 

 

 

 

 

 

 

 

 

 

 

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25 peppers (see Appendix E Fruits/Vegetables). Seasonings may include herbs such as cilantro or spices such 

26 as black pepper that are known to be contaminated with pathogens such as Salmonella, although many 

27 of these have been treated to control such microbial pathogens. (See Appendix F Spices/Herbs) 

28 Deli salads with a shelf life greater than two weeks could result in increased risk to consumers due 

29 to potential for pathogen growth (especially L. monocytogenes). Where feasible, some deli salads contain 

30 antimicrobials such as sorbate or “clean label” antimicrobials to inhibit pathogen growth for duration of 

31 the desired shelf life, which could thus reduce risk. 

32 Example 1 - potato salad 

33 Example 2 – rice, bean, and corn salad 

34 Question 1. What principles and criteria should a company apply in determining the need for and in 

35 designing an effective microbial testing program to verify that processes are effectively controlling 

36 microbial pathogens? 

37 Criteria a facility can apply to determine whether and how often to test ready-to-eat deli salads: 

Criterion/Factor Potato salad (potatoes, onions, Rice, bean, and corn salad (cooked 

celery, mayonnaise, salt, pepper, brown rice, canned black beans, 

vinegar) frozen corn, red peppers, jalapeño 

peppers, onions, cilantro, sugar, 

salt, vinegar, oil) 

A. Are pathogens Yes - Pathogens such as Yes - Pathogens such as 

associated with the Salmonella, L. monocytogenes, and Salmonella, L. monocytogenes, and 

food or ingredients? pathogenic E. coli have been pathogenic E. coli have been 

associated with raw agricultural associated with peppers and 

commodities such as potatoes, onions. L. monocytogenes has been 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Potato salad (potatoes, onions, 

celery, mayonnaise, salt, pepper, 

vinegar) 

B. Are the ingredients 

likely to be 

contaminated? 

onions, and celery, and Salmonella 

has been associated with spices 

such as black pepper (9)(Ch. 11). 

The ingredients (e.g., black pepper, 

potatoes) may also contain 

pathogenic sporeformers such as 

Clostridium botulinum, C. 

perfringens, and Bacillus cereus. 

Pathogens have not been 

associated with mayonnaise 

(commercial), salt, and vinegar. 

Yes, ingredients such as produce 

that have not received a microbial 

reduction treatment could be 

contaminated with pathogens, 

even though they have been grown 

in accordance with Good 

Agricultural Practice/produce 

Rice, bean, and corn salad (cooked 

brown rice, canned black beans, 

frozen corn, red peppers, jalapeño 

peppers, onions, cilantro, sugar, 

salt, vinegar, oil) 

associated with frozen corn. B. 

cereus has been associated with 

rice. Cyclospora has been 

associated with cilantro. Pathogens 

have not been associated with 

sugar, salt, vinegar, and oil. 

Pathogens are not associated with 

properly canned black beans. 

Yes, produce ingredients that have 

not received a microbial reduction 

treatment (e.g., peppers, onions, 

cilantro) could be contaminated 

with pathogens even though they 

have been grown in accordance 

with Good Agricultural 
 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Potato salad (potatoes, onions, 

celery, mayonnaise, salt, pepper, 

vinegar) 

safety standards to minimize 

contamination. 

C. Are there robust There is no kill step applied to the 

processing control finished product. Potatoes are 

procedures such as a cooked; black pepper will have 

kill step or other been treated (e.g., steam, ethylene 

reduction oxide, irradiation), which will kill 

methods/controls? vegetative pathogens such as 

Salmonella, but pathogenic 

sporeformers will be present. 

However, some of the ingredients 

do not have a kill step, e.g., 

chopped fresh produce, and could 

still contain pathogens such as L. 

monocytogenes and Salmonella. 

Rice, bean, and corn salad (cooked 

brown rice, canned black beans, 

frozen corn, red peppers, jalapeño 

peppers, onions, cilantro, sugar, 

salt, vinegar, oil) 

Practice/produce safety standards 

to minimize contamination. Rice is 

expected to contain B. cereus 

spores. 

There is no kill step applied to the 

finished product. Rice is cooked, 

but pathogenic sporeformers will 

be present. Canning will eliminate 

all pathogens present in black 

beans. Some of the ingredients do 

not have a kill step (e.g., chopped 

fresh produce, cilantro) and could 

still contain pathogens such as L. 

monocytogenes or Cyclospora. 

Frozen corn has been blanched but 

may have been recontaminated 

with L. monocytogenes after 

blanching. 
 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Potato salad (potatoes, onions, 

celery, mayonnaise, salt, pepper, 

vinegar) 

D.Is there a potential for 

recontamination from 

the handling or the 

environment? 

E. Does the product 

support survival or 

growth? 

Yes, the product is exposed to the 

environment during ingredient 

preparation (e.g., chopping) and 

mixing; however, sanitation 

controls verified with a robust 

environmental monitoring program 

(EMP) can reduce the potential for 

deli salads such as potato salad to 

be contaminated with microbial 

pathogens such as L. 

monocytogenes and Salmonella 

that can become established in the 

environment. 

Pathogens will survive but growth, 

if it occurs, is likely to be slow since 

product is refrigerated and most 

deli salads, including potato salad, 

are acidified to a pH of 4.5-4.9. 

Rice, bean, and corn salad (cooked 

brown rice, canned black beans, 

frozen corn, red peppers, jalapeño 

peppers, onions, cilantro, sugar, 

salt, vinegar, oil) 

Yes, the product is exposed to the 

environment during ingredient 

preparation (e.g., chopping) and 

mixing. Sanitation controls verified 

with environmental monitoring can 

reduce the potential for 

contamination with environmental 

pathogens such as L. 

monocytogenes and Salmonella. 

Rice-based salads may not be 

acidified to a pH that controls 

growth of all pathogens that may 

be present and could support 

growth of pathogens to hazardous 
 

 

 

 

 

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Criterion/Factor Potato salad (potatoes, onions, 

celery, mayonnaise, salt, pepper, 

vinegar) 

F. Is this product meant 

for higher risk 

population? 

Vinegar and mayonnaise will 

reduce the pH, which could 

prevent growth of pathogens such 

as pathogenic sporeformers, and, if 

present, Salmonella and L. 

monocytogenes. 

Challenge studies can determine 

whether there is growth or survival 

of pathogens in the formulation 

(13) 

In most instances the product is In most instances the product is 

being made for the general being made for the general 

population. However, some population. However, some 

facilities may be producing potato facilities may be producing the 

salad for hospitals or nursing salad for hospitals or nursing 

homes, where the consumers are homes, where the consumers are 

at higher risk for illness from at higher risk for illness from 

pathogens such as L. pathogens such as L. 

Rice, bean, and corn salad (cooked 

brown rice, canned black beans, 

frozen corn, red peppers, jalapeño 

peppers, onions, cilantro, sugar, 

salt, vinegar, oil) 

levels. Challenge studies can 

determine whether there is growth 

or survival of pathogens in the 

formulation (13). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Potato salad (potatoes, onions, 

celery, mayonnaise, salt, pepper, 

vinegar) 

monocytogenes. This pathogen can 

cause serious illness or death in 

susceptible consumers (in 

particular the elderly, the 

immunocompromised, and 

pregnant women). 

Rice, bean, and corn salad (cooked 

brown rice, canned black beans, 

frozen corn, red peppers, jalapeño 

peppers, onions, cilantro, sugar, 

salt, vinegar, oil) 

monocytogenes. This pathogen can 

cause serious illness or death in 

susceptible consumers (in 

particular the elderly, the 

immunocompromised, and 

pregnant women). 

G.What is the shelf life of 1-2 weeks, refrigerated 1-2 weeks, refrigerated 

the product? 

H.Will consumer handling 

and use increase or 

decrease likelihood of 

pathogen survival, 

growth, or toxin 

production and risk of 

consumer illness? 

L. monocytogenes can grow 

(although slowly) during 

refrigeration if pH is ≥4.4, therefore 

extended storage time (beyond a 

use-by date) can lead to higher 

numbers of the organism and 

increased illness risk. 

Consumers could hold potato salad 

for several hours without 

L. monocytogenes can grow 

(although slowly) during 

refrigeration if pH is ≥4.4, therefore

extended storage time (beyond a 

use-by date) can lead to higher 

numbers of the organism and 

increased illness risk. 

B. cereus can grow in cooked rice 

salads if held without refrigeration, 

but this would require several 
 

 

 

 

 

 

 

 

 

 

38 

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Criterion/Factor Potato salad (potatoes, onions, Rice, bean, and corn salad (cooked 

celery, mayonnaise, salt, pepper, brown rice, canned black beans, 

vinegar) frozen corn, red peppers, jalapeño 

peppers, onions, cilantro, sugar, 

salt, vinegar, oil) 

refrigeration (e.g., at social hours; reduced pH in the salad 

gatherings). could extend the time needed for 

growth to hazardous levels. 

Note: Most deli salads will not be Consumers could hold the salad for 

heated. A salad such as German several hours without refrigeration 

Potato Salad may be served warm, (e.g., at social gatherings). 

and heating would reduce, but not A rice, bean and corn salad may be 

eliminate, the risk from L. heated by the consumer for 

monocytogenes. palatability, which would reduce 

but not eliminate, the risk from 

pathogens such as L. 

monocytogenes. 

39 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

40 enzymes, would be an appropriate verification activity? 

41 The diversity of ingredients makes an enzyme-based test impractical. Testing pH can be an 

42 important verification activity for process control for many of these products. The pH is important in 

43 reducing growth of pathogens, especially under refrigeration, but preventing pathogen growth does not 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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control the risk of illness from pathogens such as L. monocytogenes, Salmonella, and pathogenic E. coli, 

which are relatively acid tolerant and can survive in the product. Testing of pH does not address control 

of contamination from the environment. The diversity of ingredients and large particles can result in pH 

variation throughout the product, which makes testing for pH less relevant as a verification activity for 

some deli salads. 

Question 3. Are there situations where [microbial] verification testing would not be necessary if there 

is evidence that the appropriate treatment was, in fact, applied? 

Deli salads do not receive a treatment in the final package that is lethal for the pathogens of 

concern, and the product contains ingredients that have not been subjected to a lethality process. Thus, 

there is not a treatment that can be monitored that would provide assurance that all pathogens of concern 

have been controlled. Periodic microbial testing is an appropriate verification activity in ensuring the 

safety of RTE deli salads, although routine end-product testing for pathogens is not recommended (see 

ICMSF Chapter 26)(7). Microbial testing of finished product for hygiene indicator organisms (e.g., 

coliforms, Enterobacteriaceae, generic E. coli) can be used for ongoing process control; when indicators 

suggest a potential problem, pathogen testing relevant to the product and/or ingredients may be 

considered (12). Companies should consider the risk to the intended consumer when deciding on whether 

to conduct testing for pathogens; for example, testing for pathogens should be considered when foods 

are specifically intended for highly susceptible populations (e.g., hospitals). See Question 7 on 

environmental monitoring for microbial verification testing. See the Appendix E Fruits and Vegetables 

[RTE fresh-cut vegetables] with respect to testing of produce such as onions, celery, and cilantro to verify 

supplier controls. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

As noted in Question 3, microbial testing of finished product for hygiene indicator organisms can 

be used for ongoing process control. These could include coliforms, Enterobacteriaceae, or generic E. coli 

(12). Coliforms or Enterobacteriaceae may be better indicators of process control and sanitation than E. 

coli, since they would be present in greater numbers (10). However, they are not appropriate indicators 

for environmental contamination with L. monocytogenes or Salmonella. Environmental testing for Listeria 

should be used to assess process control and sanitary conditions (12). Periodic testing of the environment 

for Salmonella may also be warranted. If deli salads are being prepared specifically for at-risk populations 

(e.g., hospitals, nursing homes), testing of the finished product should include Listeria monocytogenes. 

Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

A company should consider how robust the control measures are with respect to efficacy and 

implementation. This includes the company’s process control and sanitation control measures, as well as 

those of suppliers, when applicable. The company should also consider data from verification testing of 

product for indicator organisms, applicable supplier verification testing, and from environmental 

monitoring programs. A company could conduct more frequent testing of product initially to obtain 

baseline information; this testing could include some pathogen testing, as well as indicator organisms. 

The frequency of finished product microbiological testing can be reduced the longer the production 

process is found to be under control (See ICMSF Chapter 18; (7). Testing for pathogens should be 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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increased when verification activities indicate a problem that has the potential to result in pathogen 

contamination of the food. 

Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

the goal of verifying the adequacy of control of microbial hazards? 

Monitoring and verification of processing steps such as the cook step for certain components of 

deli salads to ensure validated process controls are appropriately implemented, combined with testing of 

the ingredients of concern (e.g., those that have not received a lethality treatment), could be an 

alternative to finished product testing. However, in many cases the ingredients of concern may have a 

short shelf life, and unless the test results can be obtained within approximately 24 hours, such testing 

may not be practical. 

Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

product testing verification activities by companies? 

A robust EMP should reduce the need for finished product testing, since one of the main 

pathogens of concern for deli salads is Listeria monocytogenes, which primarily comes from 

environmental contamination. The EMP should also periodically test for Salmonella, which is associated 

with the raw vegetables and grains and can become established in the environment. The results of 

environmental monitoring could result in the need for microbiological testing of product or ingredients 

(e.g., if a food contact surface tests positive for Listeria spp. or L. monocytogenes, product or ingredient 

testing may be part of investigative testing or root cause analysis [FDA, 2017]). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Question 8: What criteria should a company apply in determining that microbial testing results indicate 

a loss of (systemic) process control? 

What actions should a company take if test results indicate a loss of control? 

When verification testing indicates loss of process control, to what extent should verification testing be 

increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

A company should consider the finding of a pathogen in an RTE deli salad to indicate a likely loss 

of process control. In addition, the finding of indicator organisms exceeding the established limits could 

also indicate a loss of process control. In all cases, investigation is warranted. The investigation could 

indicate the need for additional testing to determine the root cause of the problem or to determine, in 

the case of indicator organisms, whether pathogen testing is warranted. 

If E. coli, coliforms, or Enterobacteriaceae exceed a defined limit investigation into the cause is 

warranted. In some cases, product testing for pathogens such as Salmonella or pathogenic E. coli may be 

warranted when limits for indicator organisms are exceeded. This may depend on the findings of a root 

cause analysis of the issue, or companies may establish a protocol for when such testing would be done 

based on the overall food safety system, the likelihood that a pathogen could be present, and the risk to 

the consumer. Corrective actions should be taken for any finding of Listeria in the environment; corrective 

actions followed by repeat positives may indicate the need for product testing for Listeria monocytogenes 

(see FDA draft guidance on Control of Listeria monocytogenes in RTE Foods FDA (20). Similarly, corrective 

actions for finding Salmonella in the environment may indicate the need for product testing for this 

pathogen. 
 

 

 

 

 

 

 

 

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Recommendations: Based on the above, we recommend that for deli salads: 

• Microbiological testing of finished product and the environment should play a role in the 

verification of control measures. 

• Periodic testing of finished product (e.g., quarterly) for pathogens (e.g., Salmonella, L. 

monocytogenes) should be conducted to verify process control. In addition, “for cause” pathogen 

testing is recommended (e.g., when a problem is detected that indicates the potential for the 

food to be contaminated with a pathogen). Routine testing for E. coli, Enterobacteriaceae or 

coliforms should be conducted more frequently (e.g., daily or weekly) than tests for pathogens. 

(See Table C-1 for microorganisms and common limits.) 

• Deli salad makers should conduct activities to verify that suppliers have implemented control 

measures to minimize the potential for pathogens to be present in those deli salad ingredients 

that have been associated with pathogens (e.g., chopped onions and celery). 

• Supplier verification activities should include microbiological testing of certain ingredients (e.g., 

chopped onions and peppers, cilantro and other ingredients that have not received a step lethal 

to the pathogens of concern) by the supplier or the deli salad manufacturer (e.g., for Salmonella 

and L. monocytogenes); the frequency of such testing should be based on an assessment of the 

likelihood of the ingredients supplied being contaminated, considering the likelihood of 

contamination of the raw material for the ingredients supplied and the control programs 

implemented by the supplier. 

• Deli salad manufacturers should implement robust environmental monitoring programs for 

Listeria spp. (and periodically for Salmonella) to ensure that the salads are not contaminated from 

the processing environment; having a robust environmental monitoring program minimizes the 

need for finished product testing. 
 

 

 

 

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155 Table C-1. Example of product testing for deli salads 

Target Microorganism Microbial Limit Recommended Action if 

Limit is Exceeded 

Comments 

Coliforms or 

Enterobacteriaceae 

<100 cfu/g Investigate reason for 

exceeding limit and 

correct. Determine if 

pathogen testing is 

warranted. 

Coliforms, 

Enterobacteriaceae, 

or E. coli are 

acceptable for 

routine testing 

(10). Only one of 

these indicators is 

needed. As noted 

above, coliforms or 

Enterobacteriaceae 

may be better 

indicator of process 

control and 

sanitation than E. 

coli, since they 

would be present in 

greater numbers. 

E. coli a <10 cfu/g Investigate reason for 

exceeding limit and 

correct. Determine if 

pathogen testing is 

warranted. 

Salmonella negative in 375 g Destroy lot. Investigate 

cause of contamination. 

Determine if other lots 

Can composite 15 

25g samples into 

one 375 g analytical 
 

 

 

 

 

 

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Target Microorganism Microbial Limit Recommended Action if 

Limit is Exceeded 

Comments 

involved. Determine 

steps to prevent 

reoccurrence. 

unit; Sample size 

should increase for 

investigation 

sampling (e.g., 60 

25g samples tested 

individually or 

composited into 4 

375 g analytical 

units 

Listeria monocytogenes Negative in 25g Destroy lot. Investigate 

cause of contamination. 

Determine if other lots 

involved. Determine 

steps to prevent 

reoccurrence. 

156 

157 B. Sandwiches 

158 Sandwiches have many combinations of ingredients, including breads, meats, cheeses, produce 

159 (e.g., lettuce, tomato), salads (e.g., chicken, egg), and condiments. Some sandwiches may be prepared 

160 for reheating for palatability prior to serving (e.g., an egg and biscuit sandwich, which can also contain 

161 meat such as sausage). In many instances the sandwiches are assembled manually, which can result in 

162 contamination. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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163 Sandwich example 1: Ham, turkey, or roast beef with bread, cheddar cheese, lettuce, tomato, 

164 mayonnaise, mustard (refrigerated) 

165 Sandwich Example 2: Sausage and egg biscuit sandwich (frozen) 

166 

167 Question 1. What principles and criteria should a company apply in determining the need for and in 

168 designing an effective microbial testing program to verify that processes are effectively controlling 

169 microbial pathogens? 

170 Criteria a facility can apply to determine whether and how often to test ready-to-eat sandwiches: 

Criterion/Factor 

Ham, turkey, or roast beef with 

bread, cheddar cheese, lettuce, 

tomato, mayonnaise, mustard 

(refrigerated) 

Sausage and egg biscuit sandwich 

(frozen) 

A. Are pathogens Meat has pathogens such as Sausage has pathogens such as 

associated with the Salmonella and L. monocytogenes Salmonella and L. monocytogenes 

food or ingredients? that are addressed through cooking 

of meat by the supplier and 

prevention of recontamination 

after cooking. Pathogenic 

sporeformers in meat (e.g., C. 

perfringens) are controlled by 

refrigeration. Salmonella in flour is 

addressed in baking bread. 

Pathogens associated with cheese 

that are addressed through cooking 

of sausage by the supplier and 

prevention of recontamination 

after cooking. Pathogenic 

sporeformers in meat (e.g., C. 

perfringens) are controlled by 

refrigeration. Salmonella and 

pathogenic E. coli potentially 

present in flour are addressed by 
 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor 

Ham, turkey, or roast beef with 

bread, cheddar cheese, lettuce, 

tomato, mayonnaise, mustard 

(refrigerated) 

Sausage and egg biscuit sandwich 

(frozen) 

are addressed in its manufacture 

through pasteurization of the milk, 

production of acids, and reduction 

of pH through microbial growth of 

starter cultures, and through aging, 

as well as through controls to 

minimize contamination with L. 

monocytogenes from the 

environment. Lettuce and tomato 

have the potential to contain 

pathogens such as Salmonella from 

the growing environment, 

addressed in part by controls 

applied during growing and 

harvesting. Condiments are not 

likely to contain pathogens. 

the supplier in baking the biscuits. 

Salmonella is associated with eggs 

(21), but the organism will be killed 

when the egg is cooked. The egg 

ingredient in the sandwich is likely 

to be made using pasteurized liquid 

whole egg, and thus will be 

subjected to two lethal processes -

pasteurization of the liquid whole 

egg at a USDA establishment and 

cooking of the liquid egg for the 

sandwich. 

B. Are the ingredients 

likely to be 

contaminated? 

Suppliers will control the hazards in 

many of the ingredients used in 

making sandwiches, such as meats 

If the sandwiches are assembled 

from pre-cooked sausage, eggs, 

and biscuits from a supplier, the 
 

 

 

 

 

 

 

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Criterion/Factor 

Ham, turkey, or roast beef with 

bread, cheddar cheese, lettuce, 

tomato, mayonnaise, mustard 

(refrigerated) 

Sausage and egg biscuit sandwich 

(frozen) 

and cheeses. Although the 

potential is relatively low, 

pathogens are considered 

reasonably likely for produce such 

as lettuce and tomatoes because 

there is no kill step. There is also 

the potential for the meats used in 

these sandwiches to be 

contaminated with L 

monocytogenes from the supplier’s 

environment (see Criterion/Factor 

D and Question 3). 

ingredients have a low potential to 

be contaminated, provided that the 

suppliers properly implement 

process controls and prevent 

recontamination from the 

environment. If the sandwich 

manufacturer prepares any of the 

components from raw ingredients 

such as raw meat, shell eggs, and 

flour, these raw ingredients should 

be considered likely to be 

contaminated. If the manufacturer 

uses pasteurized liquid whole egg, 

the potential for Salmonella to be 

present is low. 

C. Are there robust Yes, for meats, bread, and cheese, Yes, for sausage, biscuit, and egg, 

processing control but not for the lettuce and tomato but not for the assembled 

procedures such as a or for the assembled sandwich. sandwich. 

kill step or other 
 

 

 

 

 

 

 

 

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Criterion/Factor 

Ham, turkey, or roast beef with 

bread, cheddar cheese, lettuce, 

tomato, mayonnaise, mustard 

(refrigerated) 

Sausage and egg biscuit sandwich 

(frozen) 

reduction methods/ 

controls? 

D. Is there a potential for Yes, for both handling (sandwich Yes, for both handling (during 

recontamination from assembly) and from the sandwich sandwich assembly) and from the 

the handling or the manufacturing environment; environment; employee GMPs and 

environment? employee GMPs and sanitation 

controls for the environment, 

verified with an EMP, are needed 

to minimize the likelihood of 

contamination. In addition, there is 

potential for recontamination of 

the meat and cheese ingredients in 

the suppliers’ manufacturing 

environments. Some deli meats 

may receive a process in the 

package (e.g., high-pressure 

processing or a heat treatment) to 

inactivate low levels of L. 

monocytogenes present due to 

sanitation controls for the 

environment, verified with an EMP, 

are needed to minimize the 

likelihood of contamination. In 

addition, there is potential for 

recontamination of the sausage 

and biscuits ingredients in the 

suppliers’ manufacturing 

environments. 
 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor 

Ham, turkey, or roast beef with 

bread, cheddar cheese, lettuce, 

tomato, mayonnaise, mustard 

(refrigerated) 

Sausage and egg biscuit sandwich 

(frozen) 

recontamination from 

environment. 

E. Does the product 

support survival or 

growth? 

Yes. Refrigeration will slow growth 

(e.g., for L. monocytogenes) or 

prevent growth (e.g., pathogenic 

sporeformers) of pathogens. 

Cheddar cheese is a hard cheese 

that will not support growth of L. 

monocytogenes, but the organism 

will survive if the cheese is 

contaminated from the 

environment. Meat ingredients 

may contain inhibitors to growth of 

L. monocytogenes (e.g., lactate and 

diacetate) (14) 

Yes, pathogens (e.g., L 

monocytogenes, if present, and 

pathogenic sporeformers) will 

survive, although freezing will 

prevent growth. If the sandwich is 

thawed, growth of pathogens could 

occur, depending on the 

temperature. 

F. Is this product meant In most instances the product is In most instances the product is 

for higher risk being made for the general being made for the general 

population? population. However, some 

facilities may be producing 

population. However, some 

facilities may be producing 
 

 

 

 

 

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Criterion/Factor 

Ham, turkey, or roast beef with 

bread, cheddar cheese, lettuce, 

tomato, mayonnaise, mustard 

(refrigerated) 

Sausage and egg biscuit sandwich 

(frozen) 

sandwiches for hospitals or nursing 

homes, where the consumers are 

at higher risk for illness from 

pathogens such as L. 

monocytogenes. 

sandwiches for hospitals or nursing 

homes, where the consumers are 

at higher risk for illness from 

pathogens such as L. 

monocytogenes. 

G. What is the shelf life of 

the product? 

Short: 1-2 days maximum for 

ham/turkey/roast beef sandwiches 

with lettuce, tomato, and cheese. 

Thus, the time available for 

pathogen growth will be short. 

Several months when frozen, a few 

days if thawed and refrigerated. No 

growth will occur during frozen 

storage; if the product is thawed 

and held refrigerated, the time 

available for growth will be short. 

H. Will consumer handling If the sandwiches are contaminated If the consumer keeps the 

and use increase or with L. monocytogenes, and the sandwich frozen until heated and 

decrease likelihood of consumer holds the sandwiches consumed, there is no increased 

pathogen survival, under refrigeration for a day or risk. Heating the product could 

growth, or toxin more, the risk of illness from that potentially decrease the risk of 

production and risk of organism could increase. The risk illness from L. monocytogenes (if 

consumer illness? of growth from pathogenic 

sporeformers is very low, since 

present) by reducing the number of 

organisms. 
 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor 

Ham, turkey, or roast beef with 

bread, cheddar cheese, lettuce, 

tomato, mayonnaise, mustard 

(refrigerated) 

Sausage and egg biscuit sandwich 

(frozen) 

significant temperature abuse 

would have to occur, and the 

sandwich is likely to spoil and not 

be consumed. 

171 

172 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

173 enzymes, would be an appropriate verification activity? 

174 No, in particular the diversity of ingredients makes an enzyme-based test impractical. The 

175 diversity of ingredients also makes testing for pH and aw not relevant as a verification activity for most 

176 sandwiches. 

177 

178 Question 3. Are there situations where [microbial] verification testing would not be necessary if there 

179 is evidence that the appropriate treatment was, in fact, applied? 

180 There is no single treatment applied to sandwiches that can be monitored and verified as a means 

181 of demonstrating that pathogens have been controlled. Because of the short shelf life of most 

182 sandwiches, companies need to rely on supplier controls, process controls, control of employee practices, 

183 and environmental (sanitation) controls rather than microbial testing of sandwiches for verification of 

184 pathogen control. Environmental monitoring to verify sanitation controls will be needed. In these 

185 examples, there are robust process controls (lethality steps) in the manufacture of the meats (ham, 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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186 turkey, roast beef, sausage), cheese, bread, biscuit, and egg that would not need to be verified by routine 

187 microbial testing. For example, USDA FSIS has lethality requirements for a 6.5 log reduction of Salmonella 

188 in roast beef (9 CFR 318.17(a)(1)), cooked poultry products must be processed to achieve at least a 7-log 

189 reduction of Salmonella (9 CFR 381.150(a)(1)), and uncured meat patties must be processed to meet or 

190 exceed the times and temperatures listed in 9 CFR 318.23, which will achieve a 5-log lethality (15). Typical 

191 commercial processes for baking (e.g., whole wheat bread, hamburger buns) have been shown to achieve 

192 a significant reduction (e.g., >5 logs) of Salmonella (5, 6), although suppliers should provide validation 

193 information for the specific baked goods. USDA requires pasteurized egg products to be produced to be 

194 edible without further preparation to achieve food safety and to be sampled for Salmonella spp. (9 CFR 

195 590.570 and 590.580)

1 

. In general, FSIS considers a 5-log reduction of Salmonella to provide safety in 

196 products that are edible without additional preparation to achieve food safety, including egg products 

197 (17). 

198 However, many of these ingredients are likely exposed to the environment after the lethality step 

199 and, thus, there is the potential for recontamination from the environment. For example, USDA FSIS 

200 reported that the Salmonella percent positive in pasteurized egg products from 2008 to 2017 was 0.14% 

201 (although there have not been any positives in pasteurized liquid whole egg since 2012) (16). USDA FSIS 

202 also reported only one (of more than 14,000) samples of RTE meat and poultry tested positive for 

203 Salmonella in 2017, but 30 samples (0.20 %) tested positive for L. monocytogenes (18). It would be 

204 appropriate for the suppliers of the luncheon meat, sausage, pasteurized egg, and the cheese to 

205 periodically conduct product testing to verify their process control and their sanitation control measures 

1 USDA has amended the egg products inspection regulations by requiring official plants 

that process egg products to develop and implement HACCP systems and to process egg products to be 

edible without additional preparation to achieve food safety, i.e., ensure that the products are free of 

detectable pathogens (85 Federal Register 68640, October 29, 2020); minimum times and temperatures 

for pasteurization in 9 CFR 590.570 were moved to the FSIS Food Safety Guidelines for Egg Products, 

September 9, 2020 (U.S. Department of Agriculture Food Safety and Inspection Service, 2020). 
 

 

 

 

 

 

 

 

 

 

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based in part on the results of a robust EMP. It would also be appropriate for the sandwich manufacturer 

to periodically conduct testing of these ingredients as part of a supplier verification program. The 

frequency of the testing would depend on factors such as results of a supplier audit, history of supplier 

compliance, association of the ingredient with pathogen contamination and illnesses, etc. Testing of 

bread/biscuits is not warranted (provided the supplier has been verified to have appropriate process 

control and sanitation control measures verified by an EMP); recontamination of bread from the 

environment has not been the cause of foodborne outbreaks. 

Question 4. When microbial testing is an appropriate verification activity (for finished product), what 

considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

Routine finished product testing for pathogens is not practical for short shelf-life sandwiches. 

There could be benefit from enumerating indicator organisms such as coliforms, Enterobacteriaceae or 

generic E. coli to identify changes in microbiological counts that warrant investigation of process, 

sanitation, and supplier controls, as well as facility CGMP practices. Since sanitation controls are essential 

to prevent contamination from food handlers and the environment during assembly of sandwiches, 

verification of sanitation controls provides greater benefit than finished product testing. ATP swabs after 

cleaning surfaces (including utensils) provide a useful tool to verify cleaning procedures, and 

environmental testing for Listeria spp. is needed to assess whether there are insanitary conditions that 

could lead to contamination with these organisms from the environment. Likewise, since suppliers control 

the hazards in many of the components used to make sandwiches, verifying the control measures that 

suppliers have in place, including process controls and sanitation controls, also provides greater benefit 

than finished product testing of sandwiches. Verification of supplier controls on ingredients should include 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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audits and may include microbial testing reported in COAs. Out-of-specification ingredients (for those 

tested when warranted) could be diverted from use in sandwiches. Microbial testing for pathogens (e.g., 

for certain ingredients used in sandwiches) should be considered, in particular when the sandwiches are 

specifically intended for highly susceptible populations (e.g., hospitals). 

Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

Routine finished product testing for pathogens is not warranted, as discussed in Questions 3 and 

4. As noted in Question 4, enumerating indicator organisms such as coliforms, Enterobacteriaceae or 

generic E. coli could be used to indicate inadequate controls that could result in an increased risk of 

pathogens being present. The frequency of process control testing will depend on a variety of factors (see 

Main Document, Question 5). Regardless, testing more frequently will be more effective in identifying a 

loss of process control, assist in root cause analysis and in determining when control has been restored 

(3). 

Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

the goal of verifying the adequacy of control of microbial hazards? 

Supplier controls are critical; testing and COAs from suppliers (or periodic testing of ingredients 

by the receiving facility) may be appropriate in some circumstances, but may not be warranted (or may 

be limited) if a firm can verify a supplier has adequate process controls and control of environmental 

contamination verified with an EMP. For example, with respect to the meat and cheese ingredients of the 

example luncheon meat sandwich, periodic testing for L. monocytogenes before use of the ingredients 

would be appropriate, with the frequency dependent on the strength of the supplier’s control measures 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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ad supplier performance. Similarly, periodic testing for L. monocytogenes and Salmonella in the sausage 

and for Salmonella in the egg would be appropriate for the example sausage and egg biscuit sandwich. 

Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

product testing verification activities by companies? 

EMP is a key factor in not conducting or in limiting finished product testing. Because the greatest 

likelihood of pathogens being present comes from environmental contamination (assuming suppliers’ 

control programs are appropriate and properly implemented), environmental monitoring on an ongoing 

basis to verify sanitation controls provides the most relevant information on product safety. The results 

of environmental monitoring could result in the need for microbiological testing of product or ingredients 

(e.g., if a food contact surface tests positive for Listeria spp. or L. monocytogenes, product or ingredient 

testing may be part of investigative testing or root cause analysis)(20). 

Question 8: What criteria should a company apply in determining that microbial testing results indicate 

a loss of (systemic) process control? 

What actions should a company take if test results indicate a loss of control? 

When verification testing indicates loss of process control, to what extent should verification testing be 

increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

A company should consider the finding of a pathogen in an RTE sandwich or RTE ingredient for 

the sandwich to indicate a likely loss of process control. In addition, the finding of indicator organisms 

exceeding the established limits could also indicate a loss of process control. In all cases, investigation is 

warranted. The investigation could indicate the need for additional testing to determine the root cause 

of the problem or to determine, in the case of indicator organisms, whether pathogen testing is 

warranted. 
 

 

 

 

 

 

 

 

 

 

 

 

 

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If problems occur at a supplier (e.g., a meat provider has a problem with a pathogen being 

detected in RTE meat) and the supplier has taken appropriate corrective action, a company may consider 

testing that ingredient (or requiring a COA) for a period of time as a verification that the corrective actions 

have been effective. In addition, corrective actions should be taken by the sandwich manufacturer of by 

a supplier of an RTE ingredient for any finding of Listeria spp. in the environment; corrective actions 

followed by repeat positives may indicate the need for product testing for L. monocytogenes (see FDA 

draft guidance on Control of Listeria monocytogenes in RTE Foods (20). 

If testing for E. coli, Enterobacteriaceae or coliforms indicates a loss of control, testing frequency 

should be increased to assist in root cause analysis and to more quickly determine when control has been 

restored (3) 

Recommendations: Based on the above, we recommend that: 

• Microbiological testing of ingredients, the environment, and, to a limited extent, finished product 

should play a role in the verification of control measures for sandwiches. (See Table C-2 for 

microorganisms and common limits that could be applied when testing products.) 

• Sandwich makers should conduct activities to verify that suppliers have implemented control 

measures to minimize the potential for pathogens to be present in those sandwich ingredients 

that have been associated with pathogens. Supplier verification activities could include 

microbiological testing of ingredients; the frequency of such testing should be based on an 

assessment of the likelihood of the ingredients supplied being contaminated, considering the 

likelihood of contamination of the raw material for the ingredients supplied and the control 

programs implemented by the supplier. 

• Sandwich manufacturers should implement robust environmental monitoring programs for 

Listeria spp.to ensure that the sandwiches are not contaminated from the processing 
 

 

 

 

 

 

 

 

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301 environment; having a robust environmental monitoring program minimizes the need for finished 

302 product testing for environmental pathogens. 

303 • Routine finished product testing for pathogens is not recommended. Microbiological testing of 

304 finished product for indicator organisms such as E. coli, Enterobacteriaceae or coliforms rather 

305 than pathogens should be conducted to verify process and sanitation control and identify changes 

306 in microbiological counts warranting investigation. 

307 

308 Table C-2. Example of product testing for sandwiches 

Target 

Microorganism 

Microbial Limit Recommended Action if 

Limit is Exceeded 

Comments 

Coliforms or 

Enterobacteriaceae 

<100 cfu/g Investigate reason for 

exceeding limit and 

correct. Determine if 

pathogen testing is 

warranted. 

Routine testing. 

Coliforms, 

Enterobacteriaceae, or E. 

coli are acceptable for 

routine testing (10). Only 

one of these indicators is 

needed. As noted above, 

coliforms or 

Enterobacteriaceae may 

be better indicator of 

process control and 

sanitation than E. coli, 

since they would be 

E. coli <10 cfu/g Investigate reason for 

exceeding limit and 

correct. Determine if 

pathogen testing is 

warranted. 
 

 

 

 

 

 

 

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Target 

Microorganism 

Microbial Limit Recommended Action if 

Limit is Exceeded 

Comments 

present in greater 

numbers. 

Salmonella negative in 375 g Destroy lot. Investigate 

cause of contamination. 

Determine if other lots 

involved. Determine steps 

to prevent reoccurrence. 

Investigative testing as 

response to EMP that 

suggests likely 

contamination of product. 

Can composite 15 25g 

samples into one 375 g 

analytical unit; Sample 

size should increase for 

investigation sampling 

(e.g., 60 25g samples 

tested individually or 

composited into 4 375 g 

analytical units 

Listeria Negative in 25g Destroy lot. Investigate Investigative testing as 

monocytogenes cause of contamination. 

Determine if other lots 

involved. Determine steps 

to prevent reoccurrence. 

response to EMP that 

suggests likely 

contamination of product 

309 

310 C. “Heat and Eat” Entrées and Meals 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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311 Examples include macaroni and cheese, vegetable raviolis, soy/vegetable meat analogues (e.g., 

312 vegetable patties), samosas, pierogis, egg rolls, tofu vegetable pot pies; these could be refrigerated or 

313 frozen. These have been cooked and only require heating for palatability. (If fillings contain non-fully 

314 cooked ingredients, the products would be not-ready-to-eat (“Cook and Eat”); these products are outside 

315 the scope of the charge.) 

316 

317 Entrée Example 1 - Fried Vegetable Egg Roll (Refrigerated) 

318 Entrée Example 2 - Baked Tofu and Vegetable Pot Pie (Frozen) 

319 

320 Question 1. What principles and criteria should a company apply in determining the need for and in 

321 designing an effective microbial testing program to verify that processes are effectively controlling 

322 microbial pathogens? 

323 Criteria a facility can apply to determine whether and how often to test ready-to-eat “Heat 

324 and Eat” Entrées and Meals: 

Criterion/Factor Fried Vegetable Egg Roll Baked Tofu Vegetable Pot Pie 

(Refrigerated) flour (in the (Frozen) flour (wheat, rice), 

wrappers), vegetables (cabbage, vegetables (potatoes, onions, 

carrots, bean sprouts), ginger carrots, peas), butter/cream base 

(cream, salt), tofu (meat analogue), 

spices (black pepper). 

A. Are pathogens Yes - Pathogens such as Yes, pathogens such as Salmonella, 

associated with the Salmonella, L. monocytogenes, and L. monocytogenes, and pathogenic 

food or ingredients? pathogenic E. coli have been E. coli have been associated with 
 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Fried Vegetable Egg Roll 

(Refrigerated) flour (in the 

wrappers), vegetables (cabbage, 

carrots, bean sprouts), ginger 

B. Are the ingredients 

likely to be 

contaminated? 

associated with raw agricultural 

commodities such as cabbage, 

carrots, bean sprouts. Cabbage and 

carrots may also contain 

pathogenic sporeformers such as 

Clostridium botulinum, C. 

perfringens, and Bacillus cereus. 

Salmonella has been associated 

with spices such as ginger and with 

flour. 

Yes, ingredients such as produce Yes, but limited. Black pepper has 

that have not received a microbial been treated to eliminate 

reduction treatment could be Salmonella. Vegetable items are 

contaminated with pathogens, pre-cooked or blanched (4) so they 

Baked Tofu Vegetable Pot Pie 

(Frozen) flour (wheat, rice), 

vegetables (potatoes, onions, 

carrots, peas), butter/cream base 

(cream, salt), tofu (meat analogue), 

spices (black pepper). 

raw agricultural commodities such 

as potatoes, onions, carrots, peas. 

Salmonella has been associated 

with black pepper, and Salmonella 

and pathogenic E. coli with flour. 

The ingredients (e.g., black pepper, 

potatoes) may also contain 

pathogenic sporeformers such as 

Clostridium botulinum, C. 

perfringens, and Bacillus cereus. 

Butter and cream have been 

associated with Salmonella and L. 

monocytogenes. 
 

 

 

 

 

 

 

 

 

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Criterion/Factor Fried Vegetable Egg Roll 

(Refrigerated) flour (in the 

wrappers), vegetables (cabbage, 

carrots, bean sprouts), ginger 

even though they have been grown 

in accordance with Good 

Agricultural Practice/produce 

safety standards to minimize 

contamination. 

Baked Tofu Vegetable Pot Pie 

(Frozen) flour (wheat, rice), 

vegetables (potatoes, onions, 

carrots, peas), butter/cream base 

(cream, salt), tofu (meat analogue), 

spices (black pepper). 

are not likely to be contaminated, 

provided that the suppliers 

properly implement process 

controls and prevent 

recontamination from the 

environment. Flour may be 

contaminated, but the incidence is 

generally low. If the pot pie 

manufacturer prepares any of the 

components from raw ingredients 

such as raw vegetables, these raw 

ingredients could be contaminated. 

Tofu could be contaminated with 

Listeria monocytogenes, but it is 

frequently pasteurized after 

packaging to extend shelf life. 

Butter/cream base purchased is 
 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Fried Vegetable Egg Roll 

(Refrigerated) flour (in the 

wrappers), vegetables (cabbage, 

carrots, bean sprouts), ginger 

C. Are there robust 

processing control 

procedures such as a 

kill step or other 

reduction 

methods/controls? 

D.Is there a potential for 

recontamination from 

the handling or the 

environment? 

Yes, fully cooked egg rolls receive 

frying that would be a kill step (but 

the frying needs to be validated to 

ensure all components receive 

sufficient heat).(11) 

Yes, from the environment; egg roll 

is fried after assembly and sent into 

a cooling chamber where it could 

be exposed to post-process 

contamination. Sanitation controls 

for the environment, verified with 

Baked Tofu Vegetable Pot Pie 

(Frozen) flour (wheat, rice), 

vegetables (potatoes, onions, 

carrots, peas), butter/cream base 

(cream, salt), tofu (meat analogue), 

spices (black pepper). 

pasteurized so highly unlikely to be 

contaminated. 

Yes, blanching vegetables (frozen) 

occurs before preparation of the 

pot pie (and vegetables will be 

cooked in the pie during baking). 

Tofu receives a kill step during 

manufacture (11) . Fully cooked Pot 

Pies are baked within the crust by 

the manufacturer (the process 

should be validated). 

Yes, from the environment; the 

product is baked and will be 

exposed to the environment during 

cooling prior to packaging. 

Sanitation controls for the 

environment, verified with EMP for 
 

 

 

 

 

 

 

 

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Criterion/Factor Fried Vegetable Egg Roll 

(Refrigerated) flour (in the 

wrappers), vegetables (cabbage, 

carrots, bean sprouts), ginger 

EMP, are needed to minimize the 

likelihood of contamination. 

E. Does the product 

support survival or 

growth? 

F. Is this product meant 

for higher risk 

population? 

Pathogens will survive (e.g., L 

monocytogenes, if present due to 

post-process contamination, and 

pathogenic sporeformers). 

Refrigeration will slow growth (e.g., 

for L. monocytogenes) or prevent 

growth (e.g., pathogenic 

sporeformers) of pathogens. 

In most instances the product is 

being made for the general 

population. However, some 

facilities may be producing egg rolls 

for hospitals or nursing homes 

Baked Tofu Vegetable Pot Pie 

(Frozen) flour (wheat, rice), 

vegetables (potatoes, onions, 

carrots, peas), butter/cream base 

(cream, salt), tofu (meat analogue), 

spices (black pepper). 

both L. monocytogenes and 

Salmonella, are needed to 

minimize the likelihood of 

contamination. 

Pathogens (e.g., L monocytogenes 

and Salmonella, if present due to 

post-process contamination, and 

pathogenic sporeformers) will 

survive, but freezing will prevent 

growth. 

In most instances the product is 

being made for the general 

population. However, some 

facilities may be producing pot pies 

for hospitals or nursing homes 
 

 

 

 

 

 

 

 

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Criterion/Factor Fried Vegetable Egg Roll 

(Refrigerated) flour (in the 

wrappers), vegetables (cabbage, 

carrots, bean sprouts), ginger 

Baked Tofu Vegetable Pot Pie 

(Frozen) flour (wheat, rice), 

vegetables (potatoes, onions, 

carrots, peas), butter/cream base 

(cream, salt), tofu (meat analogue), 

spices (black pepper). 

where the consumers are at higher where the consumers are at higher 

risk for illness from pathogens such risk for illness from pathogens such 

as L. monocytogenes. as L. monocytogenes. 

G. What is the shelf life of 

the product? 

H.Will consumer handling 

and use increase or 

decrease risk of 

pathogen survival, 

growth, or toxin 

production? 

Short: a few days to two weeks. 

Even with a longer shelf life, 

potential for L. monocytogenes 

growth is minimal due to 

contamination being on the 

outside of the egg roll, which has a 

low water activity. 

Risk is low since these products are 

fully cooked. They will be reheated 

for eating and likely consumed 

within a short time. Potential for 

temperature abuse if left at nonrefrigerated temperatures, which 

Several months when frozen, a few 

days if thawed and refrigerated. 

Risk is low since these products are 

fully cooked. They will be reheated 

for eating and likely consumed 

within a short time. If product is 

held at room temperature, any 

surviving pathogens could grow 
 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Fried Vegetable Egg Roll Baked Tofu Vegetable Pot Pie 

(Refrigerated) flour (in the (Frozen) flour (wheat, rice), 

wrappers), vegetables (cabbage, vegetables (potatoes, onions, 

carrots, bean sprouts), ginger carrots, peas), butter/cream base 

(cream, salt), tofu (meat analogue), 

spices (black pepper). 

could result in growth of pathogens since the pH is likely >4.6 and the 

if present. water activity is above 0.95. 

325 

326 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

327 enzymes, would be an appropriate verification activity? 

328 No, in particular the diversity of ingredients makes an enzyme-based test impractical. 

329 

330 Question 3. Are there situations where [microbial] verification testing would not be necessary if there 

331 is evidence that the appropriate treatment was, in fact, applied? 

332 Yes – Fully-cooked products that are fried or baked using a validated process do not contain any 

333 uncooked ingredients. Control of the cooking process (with monitoring) and preventing recontamination 

334 through sanitation controls verified by an EMP indicate routine microbiological testing of product is not 

335 warranted (ICMSF, Ch. 26, (7). Heating of product for palatability further reduces risk of illness from 

336 consumption of these products. 

337 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Question 4. When microbial testing is an appropriate verification activity (for finished product), what 

considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

For egg rolls and pot pies that are fully cooked routine finished product testing for pathogens is 

not necessary. Environmental monitoring for Listeria spp. and for Salmonella is recommended (7, 9). 

Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

For fully cooked, heat-and-eat meals, finished product testing for pathogens is not needed except 

for investigative testing if EMP results suggest there is a loss of control. However, routine testing for heatsensitive indicator organisms is useful for verification that the cooking process was effective; if 

enumeration limits are exceeded, investigation and corrective actions are needed. 

Question 6: Are there situations in which testing [of food] at sites other than the end of the process can 

achieve the goal of verifying the adequacy of control of microbial hazards? 

For a product with a validated lethality (cooking) process, neither in-process or finished product 

microbial testing for pathogens is useful as a routine verification activity for product. However, other 

monitoring activities of the lethality process (e.g., oven or product internal temperature, time) provides 

more assurance of safety than microbiological testing of the food. If the food is exposed to the 

environment after the process, as with egg rolls and baked pot pies, an EMP is critical. 

Question 7: What impact should [does] environmental monitoring have on frequency and extent of 

product testing verification activities by companies? 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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A robust EMP is key factor in not conducting finished product testing of a fully cooked product 

that is exposed to the environment. If EMP results suggest there is a loss of control, investigative testing 

of finished product may be part of a root cause analysis. 

Question 8: What criteria should a company apply in determining that microbial testing results indicate 

a loss of (systemic) process control? 

What actions should a company take if test results indicate a loss of control? 

When verification testing indicates loss of process control, to what extent should verification testing be 

increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

Cook step monitoring and an EMP will provide evidence of control for a fully cooked product. If 

either process or EMP monitoring suggest limits were not met (loss of control), investigative microbial 

testing for heat-sensitive indicator organisms (e.g., >10 CFU/g E. coli) in the finished product, and 

comparison with adjacent/similar lots could identify a root cause of the problem and direct corrective 

actions. If microbial test results confirm loss of process control, investigative testing for pathogens may 

be appropriate. The presence of indicator organisms alone, without evidence of insufficient cooking or 

pathogen survival, is not used to determine release of product. Investigation and corrective actions 

should be taken for any finding of Listeria or Salmonella in the environment; repeat positives may indicate 

the need for product testing for Listeria monocytogenes (see FDA draft guidance on Control of Listeria 

monocytogenes in RTE Foods (20) or for Salmonella (19). Verification testing can be scaled back when 

root cause has been identified and corrected and microbial testing confirms correction. 

Recommendations: Based on the above, we recommend that: 
 

 

 

 

 

 

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1. Validation of the cook step and monitoring of parameters that demonstrate control are needed 

to ensure appropriate log reduction is achieved for vegetative pathogens that may be present in 

the ingredients. 

2. Because there is a validated kill step for the both the refrigerated vegetable egg roll and frozen 

tofu vegetable pot pie, no finished product testing is needed when process monitoring indicates 

the process is under control and an EMP program is in place that indicates sanitation controls are 

effective in preventing contamination from the environment. 

3. The results of environmental monitoring could result in the need for microbiological testing of 

product or ingredients (e.g., if a food contact surface tests positive for Listeria spp. or L. 

monocytogenes, product or ingredient testing may be part of investigative testing or root cause 

analysis (20). Similarly, environmental monitoring indicating the presence of Salmonella could 

suggest the need to test product for Salmonella (19). 
 

 

 

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REFERENCES 

1. Alberta Health Services. 2011. Microbial guidelines for ready-to-eat foods. 

https://www.albertahealthservices.ca/assets/wf/eph/wf-eh-microbial-guidelines-for-ready-to-eatfoods.pdf, Accessed February 7, 2021. 

2. Australia New Zealand Food Authority. 2014. Australia New Zealand Food Standards Code -

Standard 1.6.1 - Microbiological Limits for Food. http://www.comlaw.gov.au/Series/F2008B00630, 

Accessed January 12, 2021. 

3. Buchanan, R., and D. W. Schaffner. 2015. FSMA: Testing as a tool for verifying preventive 

controls. Food Prot. Trends. 35:228-237. 

4. Ceylan, E., W. McMahon, and D. M. Garren. 2017. Thermal inactivation of Listeria 

monocytogenes and Salmonella during water and steam blanching of vegetables. J. Food Prot. 80:1550-

1556. 

5. Channaiah, L. H., E. S. Holmgren, M. Michael, N. J. Sevart, D. Milke, C. L. Schwan, M. Krug, A. 

Wilder, R. K. Phebus, H. Thippareddi, and G. Milliken. 2016. Validation of baking to control Salmonella 

serovars in hamburger bun manufacturing, and evaluation of Enterococcus faecium ATCC 8459 and 

Saccharomyces cerevisiae as nonpathogenic surrogate indicators. J. Food Prot. 79:544-552. 

6. Channaiah, L. H., M. Michael, J. C. Acuff, R. K. Phebus, H. Thippareddi, and G. Milliken. 2019. 

Evaluation of thermal inactivation parameters of Salmonella in whole wheat multigrain bread. Food 

Microbiol. 82:334-341. 

7. International Commission on Microbiological Specifications for Foods (ICMSF). 2011. 

Microorganisms in Foods 8: Use of Data for Assessing Process Control and Product Acceptance. Springer, 

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8. International Commission on Microbiological Specifications for Foods (ICMSF). 2018. 

Microorganisms in Foods 7: Microbiological Testing in Food Safety Management. 2nd edition. Springer 

International Publishing, New York. 

9. International Commission on Microbiological Specifications for Foods. 2005. Microorganisms in 

Foods 6: Microbial ecology of food commodities, 2nd ed. Kluwer Academic/Plenum Publishers, New 

York. 

10. Kornacki, J. I., J. B. Gurtler, and B. A. Stawick. 2013. Enterobacteriaceae, Coliforms, and 

Escherichia coli as Quality and Safety Indicators, Updated September 2013. In Y.S. S. Doores, M. 

L.Tortorello (ed.), Compendium of Methods for the Microbiological Examination of Foods American 

Public Health Association, http://ajph.aphapublications.org/doi/book/10.2105/MBEF.0222. 

11. McHugh, T. 2016. How Tofu Is Processed. Food Technology. 70:72-74. 

12. National Advisory Committee for Microbiological Criteria for Foods. 2018. Response to 

questions posed by the Department of Defense regarding microbiological criteria as indicators of 

process control or insanitary conditions. J. Food Prot. 81:115-141. 

13. National Advisory Committee on Microbiological Criteria for Foods. 2010. Parameters for 

determining inoculated pack/challenge study protocols. J Food Prot. 73:140-202. 

14. U. S. Department of Agriculture Food Safety Inspection Service. 2014. Compliance guidelines to 

control Listeria monocytogenes in post-lethality exposed ready-to-eat meat and poultry products. 

Updated January 2014. https://www.fsis.usda.gov/guidelines/2014-0001, Accessed April 26, 2021. 

15. U.S. Department of Agriculture Food Safety and Inspection Service. 2017. FSIS Salmonella 

Compliance Guidelines for Small and Very Small Meat and Poultry Establishments that Produce Readyto-Eat (RTE) Products and Revised Appendix A June 2017. https://www.fsis.usda.gov/guidelines/2017-

0008, Accessed April 26, 2021. 
 

 

 

 

 

 

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16. U.S. Department of Agriculture Food Safety and Inspection Service. 2018. FSIS Microbiological 

Testing Program for Pasteurized Egg Products, Calendar Years 1995–2017. 

https://www.fsis.usda.gov/science-data/data-sets-visualizations/laboratory-sampling-data/egg-producttesting-years-1995-2017, Accessed April 26, 2021. 

17. U.S. Department of Agriculture Food Safety and Inspection Service. 2020. FSIS Food safety 

guidelines for egg products. https://www.fsis.usda.gov/guidelines/2020-0005, Accessed April 26, 2021. 

18. U.S. Department of Agriculture Food Safety and Inspection Service. 2020. FSIS Microbiological 

Testing Program for Ready-to-Eat (RTE) Meat and Poultry Products. https://www.fsis.usda.gov/sciencedata/data-sets-visualizations/microbiology/microbiological-testing-program-rte-meat-and-3, Access 

April 26, 2021. 

19. U.S. Food and Drug Adminstration. 2012. Guidance for Industry testing for Salmonella species in 

human foods and direct-human-contact animal foods. https://www.fda.gov/media/83177/download, 

Accessed April 26, 2021. 

20. U.S. Food and Drug Adminstration. 2017. Control of Listeria monocytogenes in Ready-To-Eat 

Foods: Guidance for Industry. https://www.fda.gov/files/food/published/Draft-Guidance-for-Industry--

Control-of-Listeria-monocytogenes-in-Ready-To-Eat-Foods-%28PDF%29.pdf, Accessed April 26, 2021. 

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for Salmonella enteritidis in pasteurized liquid eggs. Int J Food Microbiol. 36:111-125. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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APPENDIX D - CATEGORY: NUTS (INCLUDING TREE NUTS AND PEANUTS) AND NUT/SEED PRODUCTS 

Nuts are defined as “low-moisture, one-seeded fruit, usually enclosed by a rigid outer casing or 

shell” and are divided into tree nuts or ground nuts. Nuts are grown around the world and global sourcing 

is common (21). Tree nuts include almonds, hazelnuts (filberts), pistachios, Brazil nuts, pecans, coconuts, 

macadamias, chestnuts, pine nuts and walnuts, while ground nuts generally refer to peanuts (21). 

Processed products made from nuts and seeds include nut butters such as peanut butter and sunflower 

butter, nut “milks”, and nut and seed “cheeses” and spreads. 

The hazards associated with peanuts and tree nuts are determined by the environment in which 

they are grown, harvested, shelled/hulled, cleaned, sorted, processed, packaged, and stored. 

Hazards associated with processed products made from nuts and seeds are determined by the 

hazards associated with ingredients, lethality process associated with manufacture and risk for exposure 

to the process environment post-lethality treatment. 

Four categories of raw and processed nut commodities or processed products made from nuts 

are considered in this evaluation of the utility and necessity for industry to test ready-to-eat (RTE) foods 

for pathogens and whether any microbiological testing is appropriate to verify pathogen control. 

1. Ready-to-eat (RTE) nuts not processed for lethality (e.g., chopped untreated tree nuts) 

2. RTE nuts processed for lethality (e.g., roasted tree nuts, roasted peanuts) 

3. RTE nut products processed for lethality (e.g., almond milk, coconut milk, nut (cashew) cheese) 

4. RTE nut/seed butters not processed for lethality beyond initial nut processing (e.g., peanut 

butter, sunflower butter) 

1. Ready-to-eat nuts not processed for lethality 

Some tree nuts are covered by FDA’s rule “Standards for Growing, Harvesting, Packing, and 

Holding of Produce for Human Consumption” (21 CFR Part 112), which sets food safety standards for farms 

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to follow in an effort to minimize the risks of microbiological contamination that may occur during the 

production of covered produce (39). Tree nuts that are covered by the Produce Safety rule (PSR) include 

pistachios, macadamia nuts, pine nuts, and walnuts. Other raw tree nuts (hazelnuts, pecans, cashews) 

and peanuts are excluded from the rule as they are considered “rarely consumed raw” (RCR) (21CFR Part 

112.2(a)(40)). While almonds are not exempt from the PSR, the FDA has stated their intent to not enforce 

the PSR requirements for raw almonds (38). Raw almonds have been associated with salmonellosis 

outbreaks (13, 19); however, USDA regulation requires almonds for North America to be treated to 

mitigate the hazard prior to sale. 

If nuts are subjected to manufacturing/processing activities not permitted under the farm 

definition, such as roasting, blanching, chopping, dicing and grinding, these activities are regulated by the 

FDA’s rule “Current Good Manufacturing Practice, Hazard Analysis, and Risk-Based Preventive Controls 

for Human Food” (the CGMP & PC rule; 21 CFR Part 117) unless an exemption applies (33). 

Nuts that are classified as rarely consumed raw are those that FDA determined are almost always 

eaten only after being cooked and are included in an exhaustive list at 21 CFR 112.2(a)(35) Hazelnuts, 

cashews, pecans, and peanuts are exempt from the produce safety regulation because heat treatment in 

some form can adequately reduce the presence pathogens. These types of nuts are not considered in 

Category 1 of this assessment. 

Category 1 of RTE raw nuts considers only chopped tree nuts and shelled whole nuts not 

processed for lethality that are manufactured, processed, packed, or held in a facility covered by the 

CGMP & PC rule unless an exemption applies. Although not chopped, some whole shelled nuts not 

processed for lethality would fall under the CGMP and PC rule. As an example, RTE whole shelled walnuts 

not processed for lethality packaged in a facility conducting other manufacturing/processing activities 

(e.g., roasting or glazing of walnuts) would be covered by the CGMP & PC rule. Producers of RTE chopped 

raw tree nuts and some types of whole RTE nuts will implement preventive controls to significantly 

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minimize or prevent hazards to provide assurances that the RTE nuts manufactured, processed, packed, 

or held in their facility will not be adulterated under section 402 of the Federal Food, Drug, and Cosmetic 

Act. These preventive controls include sanitation controls and a supply-chain program. As part of the 

supply-chain program, suppliers (i.e., farms growing, harvesting, packing, and holding the nuts) may be 

annually audited related to their compliance with the produce safety regulation. 

Question 1. What principles and criteria should a company apply in determining the need for and in 

designing an effective microbial testing program to verify that processes are effectively controlling 

microbial pathogens? 

A hazard analysis with implementation of appropriate controls is required, considering (1) 

possible microbial hazards, (2) likelihood of occurrence, (3) available processing control procedures such 

as a kill step or other reduction methods/controls, (4) potential for inherent contamination or 

recontamination after processing from handling or the environment, (5) survival (persistence) or growth 

on the product, (6) intended consumer, (7) shelf life of the product, and (8) steps in the process where 

testing would be appropriate to verify food safety controls. 

1. Are pathogens associated with the food or ingredients? 

Pathogens are associated with raw nuts not processed for lethality, including Salmonella, Shiga 

toxin-producing E. coli and Listeria monocytogenes (19, 41). Contamination of outer shells begins at 

harvest where nuts may be shaken to the ground. Direct contact with contaminated soil during harvest 

provides an opportunity for introduction of foodborne pathogens, e.g., to walnuts (1). Salmonella 

Enteritidis PT 30, E. coli O157:H7, and L. monocytogenes are capable of long-term survival on the surface 

of in-shell walnuts (1, 14). Salmonella can persist on in-shell pistachios in storage silos for up to four 

months (17). 

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In 2010, walnuts were recalled (without illness) by a company after Salmonella was detected in 

walnut halves and pieces sold to another nut company (41). Salmonella was detected in pistachio nuts 

and walnuts (11, 14), E. coli O157:H7 was found in walnuts for sale at retail markets in the U.S.(42). 

Outbreaks have been associated with pistachio nuts contaminated with Salmonella in 2009, 2016 

and 2018 (42). The 2016 outbreak was linked to the consumption of roasted pistachios produced by one 

company (11, 34). However, the outbreak strains of Salmonella Montevideo and Salmonella Senftenberg 

were also isolated from samples of raw pistachios from the farm where the pistachios were grown. 

Walnuts were implicated in a 2011 outbreak of E. coli O157:H7 in Canada (41). 

2. Are the ingredients likely to be contaminated? 

Yes. There is a risk for microbiological contamination during the growing, harvesting, packing, and 

holding of raw nuts not processed for lethality (21). 

Nuts that are harvested off the ground without mats are more likely to be contaminated with 

pathogens inherent to the soil in which they lay. Persistence through storage, packing, and holding 

continues into the retail market (42). 

3. Are there robust processing control procedures such as a kill step or other reduction methods/controls? 

No. These nut products are raw RTE foods that are not processed for lethality. Macadamia nuts, walnuts, 

and pistachios do not commonly receive a microbial reduction treatment prior to sale either whole or 

chopped. 

Control is based on the expectation that processers beyond the grower are compliant with Sanitation and 

Supply Chain Programs under the Preventive Controls Rule (21 CFR Part 117) and that growers that supply 

the raw unprocessed nuts are compliant with the Produce Safety Rule (21 CFR Part 112) and GAPs. 

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4. Is there a potential for inherent contamination or recontamination after processing from handling or 

the environment? 

Yes. The initial microbial flora of harvested nuts will include pathogens from the 

equipment/personnel used in harvesting, transportation, and storage. Shelled or unshelled dried raw 

nuts are stored refrigerated (40C) or frozen (-180C). However, pathogens such as Salmonella are not 

eliminated during refrigeration, freezing, or drying. Tree nuts may be submerged in water to remove 

debris, soften the shell (e.g., pecans), or remove floating/damaged nuts, then de-shelled physically, a 

process that may be facilitated by water sprayers (16). Contaminated water may also be a source of 

pathogens contaminating nuts. Tree nuts that are de-shelled dry can produce dust that can spread 

pathogens (16). 

5. Does the product support survival or growth? 

All nuts are artificially, or sun and air dried after harvesting. Immediate drying upon harvest 

restricts outgrowth of mold and vegetative pathogens but not their persistence. 

6. Is this product meant for higher risk population? 

In most instances, the product is made for consumption by the general population. 

7. What is the shelf life of the product? 

Months to years. Tree nuts can be stored for days to months before processing, making moisture 

control a necessity to prevent bacterial/fungal outgrowth (16). 

8. Would consumer handling and use be likely to increase or decrease risk? 

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119 (a) Heating for palatability (b) Holding a frozen food under refrigeration (c) Holding a refrigerated food 

120 beyond the use-by date? 

121 If a thermal process was applied by the consumer for palatability, then the inherent pathogen risk 

122 posed by vegetative pathogens might be mitigated to some extent depending on the process 

123 (time/temperature), but the heating might not fully eliminate the pathogen, depending on the number 

124 present. 

125 

126 Example 1: Chopped walnuts shelled, sized and packaged in a facility that also roasts nuts. 

127 Example 2: Whole shelled macadamia nuts packaged in a facility that also dices, roasts, and seasons 

128 macadamias. 

129 

130 Table D-1. Ready-to-eat nuts not processed for lethality – Examples. 

Criterion/Factor Example 1: Chopped walnuts 

shelled, sized and packaged in a 

facility that also roasts nuts. 

Example 2: Whole shelled 

macadamia nuts packaged in a 

facility that also dices, roasts, and 

seasons macadamias. 

Nuts (in hulls) are grown on a farm 

(orchards) and mechanically 

harvested by shaking the trees. The 

nuts are pushed into windrows and 

mechanically picked up from the 

orchard floor. The nuts are passed 

through a huller (wet 

Macadamias are grown on a farm 

with an adjacent husking 

operation. Nuts fall naturally to 

the orchard floor and are either 

mechanically or hand-collected 

then husked. Nuts are then 

delivered to the processing facility 

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Criterion/Factor Example 1: Chopped walnuts 

shelled, sized and packaged in a 

facility that also roasts nuts. 

Example 2: Whole shelled 

macadamia nuts packaged in a 

facility that also dices, roasts, and 

seasons macadamias. 

scrubber)/dryer, washed and dried 

to 8% moisture in a gas dryer. The inshell raw walnuts are then delivered 

to the downstream processor used in 

this example. At the processor, 

whole walnuts are sized, cracked to 

remove the outer shell, kernels are 

sized, shell and foreign material is 

mechanically blown from the 

kernels, pieces are sized into small 

pieces, hand sorted and packaged. 

where they are dried in gas dryers, 

shelled, and packaged. 

A. Are pathogens Yes, an inherent risk due to the raw Yes, an inherent risk due to the 

associated with the nature of the ingredient. Salmonella raw nature of the ingredient. 

food or ingredients? and E. coli O157:H7 were found in 

walnuts sold in retail markets in the 

U.S. (41). Long-term survival of L. 

monocytogenes on the surface of inshell walnuts can occur (1, 14). 

Salmonella can persist on in-shell 

tree nuts for extended periods of 

storage (20). Salmonella 

prevalence in macadamia nuts 

collected at retail was 4.20% (42). 

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Criterion/Factor Example 1: Chopped walnuts 

shelled, sized and packaged in a 

facility that also roasts nuts. 

Example 2: Whole shelled 

macadamia nuts packaged in a 

facility that also dices, roasts, and 

seasons macadamias. 

B. Are the ingredients There is an inherent risk for There is an inherent risk for 

likely to be microbiological contamination microbiological contamination 

contaminated? during the growing, harvesting, and 

holding of raw walnuts. Pathogens 

from the orchard floor, equipment 

used in harvesting, transportation, 

and storage are likely. 

during the growing, harvesting, 

and holding of raw macadamia 

nuts. Pathogens from the orchard 

floor, equipment used in 

harvesting, transportation, and 

storage are likely. 

C. Are there robust 

processing control 

procedures such as a 

kill step or other 

reduction 

methods/controls? 

No 

Walnuts do not require a microbial 

reduction treatment prior to sale 

either whole or chopped. 

The facility packaging raw walnuts 

should establish and implement a 

supply-chain program that requires 

its suppliers (i.e., growers) to comply 

with the Produce Safety Rule (21 CFR 

Part 112)(39) to significantly 

No 

Macadamias do not require 

microbial reduction treatment 

prior to sale either whole or 

chopped. 

The facility packaging raw 

macadamia nuts should establish 

and implement a supply-chain 

program that requires its suppliers 

(i.e., growers) to comply with the 

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Criterion/Factor Example 1: Chopped walnuts 

shelled, sized and packaged in a 

facility that also roasts nuts. 

Example 2: Whole shelled 

macadamia nuts packaged in a 

facility that also dices, roasts, and 

seasons macadamias. 

minimize pathogens on the incoming 

product. 

Produce Safety Rule (21 CFR Part 

112)(39) to significantly minimize 

pathogens on the incoming 

product. 

D.Is there a potential Yes Yes 

for recontamination Raw walnuts in shell, with an Once harvested, the outer hull is 

from the handling or inherent potential for pathogen removed mechanically within 24 -

the environment? contamination, are received into the 

facility. The process area and 

process equipment are likely to be 

contaminated from the primary 

ingredient. In addition, the facility 

itself becomes a secondary source of 

contamination. Inadequate 

sanitation could lead to harborage 

issues with the potential to 

contaminate product as it is 

48 h. The nuts are dried to a stable 

moisture level and separated by 

size. Raw macadamia nuts 

entering the facility have an 

inherent potential for pathogen 

contamination. The husking, 

drying and packaging areas and 

equipment have the potential to 

be contaminated if adequate 

controls are not in place. The 

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Criterion/Factor Example 1: Chopped walnuts 

shelled, sized and packaged in a 

facility that also roasts nuts. 

Example 2: Whole shelled 

macadamia nuts packaged in a 

facility that also dices, roasts, and 

seasons macadamias. 

processed and packaged. 

Accordingly, the facility should 

establish a sanitation program to 

significantly minimize or prevent 

biological hazards in the areas in 

which RTE walnuts are exposed to 

the environment before packaging. 

Due to the increased risk of crosscontamination with the chopping 

equipment, the facility should 

establish and implement a robust 

environmental monitoring program 

to verify its sanitation program in 

those areas. 

facility itself can be a secondary 

source of contamination; 

inadequate sanitation could lead to 

harborage issues with the potential 

to contaminate product as it is 

packaged. Accordingly, the facility 

should establish a sanitation 

program to significantly minimize 

or prevent biological hazards in the 

areas in which RTE macadamias are 

exposed to the environment 

before packaging. 

E. Does the product 

support survival or 

growth? 

The nuts are dried after harvesting 

and hulling. Immediate drying upon 

harvest restricts outgrowth of mold 

and vegetative pathogens but not 

their persistence. 

The nuts are dried after harvesting 

and husking. Immediate drying 

upon harvest restricts outgrowth 

of mold and vegetative pathogens 

but not their persistence. 

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Criterion/Factor Example 1: Chopped walnuts 

shelled, sized and packaged in a 

facility that also roasts nuts. 

Example 2: Whole shelled 

macadamia nuts packaged in a 

facility that also dices, roasts, and 

seasons macadamias. 

F. Is this product meant 

for higher risk 

population? 

The product is made for 

consumption by the general 

population. 

The product is made for 

consumption by the general 

population. 

G.What is the shelf life 

of the product? 

3 months at 20°C, 1 year at 0°C to 

4°C. (5). 

-10°C /24 months, 0°C - 10°C/12 

months, 20°C/5 months (5). 

H.Will consumer The risk for outgrowth may be Risk may be increased if added to a 

handling and use increased if the product is not kept product with a water activity that 

increase or decrease dry during storage and condensate is allows outgrowth. 

likelihood of allowed to form or if added to a Pathogen risk is reduced if cooked 

pathogen survival, product with a water activity that or baked. 

growth, or toxin allows outgrowth. 

production and risk Pathogen risk is reduced if cooked or 

of consumer illness? baked. 

131 

132 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

133 enzymes, would be an appropriate verification activity? 

134 None known. 

135 

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Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

evidence that the appropriate treatment was, in fact, applied? 

No. Verification testing for Salmonella and Listeria is routinely performed at some level by 

industry. Note: A hold, test and release program could be appropriate for this category of product and 

might be considered by some to be a type of “preventive control,” but it is more appropriately considered 

verification of all the control measures applied to that point. 

Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

specific indicator organism) and type of test (e.g., presence/absence or enumeration)? What are 

appropriate indicator microorganisms for verifying processes adequately control pathogens? 

Finished product should be tested. Selection of pathogen targets is based on prevalence studies 

and recall/outbreak information and would include Salmonella and Listeria. Testing treenuts for generic 

E. coli is also a measure of adulteration with filth (Table D-2). Note: Because these are untreated nuts 

that are RTE, a greater reliance on verification testing would be expected and verification testing would 

occur at a greater frequency in comparison to treated nuts. 

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Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

1. Level of implementation and adherence to the Produce Safety Rules/GAPs or Preventive Controls 

Rule. 

2. Efficacy of Sanitation programs - including studies to determine frequency of sanitation and length 

of runs. 

3. Environmental control - conduct environmental "deep dives" to assess where and how often 

pathogens are found and detect harborage sites. 

4. Control of water and dust. 

Greater adherence to effective programs such as these can reduce the amount of verification testing 

needed. 

Question 6: Are there situations in which testing at sites other than the end of the process can achieve the 

goal of verifying the adequacy of control of microbial hazards? 

The entire process line has an elevated risk for pathogen contamination in the absence of process 

preventive controls. While sanitation controls can reduce the hazard at specific sites where cleaning and 

sanitation activities are conducted, contamination inherent to the raw nuts can persist and can amplify. 

The entire process is represented by end product testing of product in a final package or product sampled 

while filling bulk containers. Finished product samples are, in effect, one large “swab” of the entire 

process and are the most appropriate type of samples to verify the adequacy of any controls that are 

applied in the absence of a process preventive control. Finished product testing for Salmonella, L. 

monocytogenes and generic E. coli is recommended. Additional points of verification will not negate the 

need for finished product testing, however, testing product for generic E. coli and coliforms or 

Enterobacteriaceae at start-up and from samples taken along the process in husking, drying, chopping 

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and packaging areas could identify harborage sites (buildup of biofilms, water ingress, growth points) 

where pathogens could proliferate. Their removal would reduce the overall level of process 

contamination. 

Question 7: What impact does environmental monitoring have on frequency and extent of product testing 

verification activities by companies? 

Environmental monitoring for pathogens will verify the effectiveness of sanitation/hygiene 

programs that control build up and harborage sites. However, environmental monitoring will not diminish 

the need for lot-by-lot finished product testing. Minimally, monitoring for Salmonella and Listeria spp. as 

an indicator for L. monocytogenes on Zone 2 and 3 surfaces should be conducted (28). 

Microbiological limits for hygiene verification testing of cleaned/sanitized product contact 

surfaces in a raw nut processing facility should be established and tracked. Recommended indicator 

organisms include aerobic plate counts, coliforms, generic E. coli and/or Enterobacteriaceae. 

Question 8: What criteria should a company apply in determining that microbial testing results indicate a 

loss of (systemic) process control? What actions should a company take if test results indicate a loss of 

control? When verification testing indicates loss of process control, to what extent should verification 

testing be increased, how far upstream and downstream should it go, and when and how should it be 

scaled back? 

A food safety system and the manufacturing process managed by that system are in control when, 

within the limits of a stable and predictable process variation, all food safety hazards are controlled 

to an acceptable level. This requires the development of measurable attributes that indicate whether a 

process maintains or surpasses an acceptable degree of hazard control or falls below that level (21). 

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Producers of RTE chopped raw tree nuts and some types of whole RTE nuts rely on preventive 

controls that include sanitation controls and a supply-chain program. Control is based on the expectation 

that processers beyond the grower are compliant with Sanitation and Supply Chain Programs under the 

Preventive Controls Rule (21 CFR Part 117) and that growers that supply the raw unprocessed nuts are 

compliant with the Produce Safety Rule (21 CFR Part 112) and GAPs. Finished product testing is conducted 

to verify that sanitation controls are in place and effective within the manufacturing facility. Product 

testing for Salmonella, L. monocytogenes and generic E. coli provides highly relevant verification data and 

is appropriate for the level of risk associated with the raw nuts. Loss of control would be indicated by the 

finding of a positive pathogen result. When a pathogen is detected from a sample taken at the end of the 

production line, the recommended action is to reject the lot of raw nuts represented by the sample, 

especially when the food will not receive further processing using a validated kill step (29). Contaminated 

nuts may be reconditioned with a kill step. 

The repeated finding of an indicator organism such as generic E. coli above a threshold level can 

also indicate a loss of sanitation control, although actions taken would follow a tiered approach based on 

numbers and frequency of occurrence (see Table D-2). 

The types of actions that companies may take in the case of loss of control indicated by finished 

product verification results depending on the seriousness of the risk include: 

1. Verification of the sanitation program 

2. Increased verification testing frequency 

3. Stopping the processing line until a root analysis is completed 

4. Investigation of the source of contamination 

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222 Table D-2. Microbiological Limits for Ready-to-eat nuts not processed for lethality (29). 

Target Microorganism Microbiological Limit Recommended Action 

if Limit is Exceeded 

Comments 

E. coli (generic) <0.36 MPN/g Investigate, implement 

corrective action 

If 2 of 10 samples are 

>0.36 MPN/g, follow 

CPG Sec 570.450 (36) 

Listeria monocytogenes Negative in 25 g 

Reject. Investigate and 

implement corrective 

action 

Salmonella Negative in two 375 g 

samples 

Reject. Investigate and 

implement corrective 

action 

Two 375 g analytical 

units derived from 30 x 

25 g samples 

223 

224 

225 

226 

227 

228 

2. Ready-to-eat nuts and seeds processed for lethality 

Examples: roasted tree nuts, roasted peanuts (whole or chopped), almonds treated by propylene oxide 

(PPO), blanched almonds, salted and roasted inshell sunflower seeds, salted and roasted pumpkin seeds, 

pistachios treated by steam, roasted pecans, roasted cashews. 

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Question 1. What principles and criteria should a company apply in determining the need for and in 

designing an effective microbial testing program to verify that processes are effectively controlling 

microbial pathogens? 

1. Are pathogens associated with the food or ingredients? 

Yes. Salmonella is a pathogen that can survive for long periods of time in low-moisture food 

products or ingredients. This pathogen is widely recognized as the pertinent pathogen in low moisture 

food such as nut and seed commodities because of outbreaks associated with these foods. Processed nut 

and seed commodities have been associated with pathogens such as Salmonella, shiga toxin-producing E. 

coli and Listeria monocytogenes. Since 2017, dry roasted macadamia nuts, dry roasted pistachios, and 

roasted cashews have been recalled due to known or suspected L. monocytogenes, Salmonella, and L. 

monocytogenes contamination, respectively (42). A multistate outbreak of Salmonella Montevideo and 

Salmonella Senftenberg infections was linked to the consumption of roasted pistachios in 2016 (34). To 

date, there are no reported outbreaks of listeriosis or enterohemorrhagic E. coli infections linked to 

processed nut and seed commodities. 

2. Are the ingredients likely to be contaminated? (21) 

Extensive surveys have been conducted to determine prevalence and levels of pathogens on raw 

nuts, such as inshell walnuts (14), inshell pistachios (17), shelled almonds (27), inshell pecans (2), and 

shelled peanuts (4). Facilities that are implementing a process preventive control for biological hazards on 

their RTE nuts generally would not be relying on supply-chain preventive controls for biological hazards 

such as Salmonella, since they are processing the nuts to control the hazards. Facilities that are not 

processing their nuts with a process that significantly minimizes biological hazards may have established 

a supply-chain program to control for biological hazards such as Salmonella. An example of this type of 

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facility is one that purchases bulk packed nuts that have previously been subject to a kill step for packaging 

in RTE form. 

3. Are there robust processing control procedures such as a kill step or other reduction methods/controls? 

Yes. There are several forms of processing that nuts can be subjected to as processes to 

significantly minimize or prevent biological hazards. Such processes would likely be process controls and 

include oil roasting, dry roasting, toasting, propylene oxide treatment, steam treatment, and blanching. 

4. Is there a potential for inherent contamination or recontamination after processing from handling or 

the environment? 

Yes. RTE processed nuts may be exposed to the environment after processing with a kill step. In 

these instances, finished product testing should be considered, particularly in operations that may 

operate for extended periods of time (e.g., a week or more) between cleaning and sanitizing activities. 

Facilities subject to the requirements of subpart C of the CGMP and Preventive Controls for Human Food 

rule (21 CFR part 117) must include in their hazard analysis an evaluation of environmental pathogens 

whenever a ready-to-eat food is exposed to the environment prior to packaging and the packaged food 

does not receive a treatment or otherwise include a control measure that would significantly minimize 

the pathogen. 

5. Does the product support survival or growth? 

Pathogens will survive but growth is prevented by low water activities. Numerous studies have 

evaluated the ability of Salmonella, E. coli O157:H7, and L. monocytogenes to persist on various tree nuts 

and peanuts for months at various storage temperatures (20). Salmonella is widely recognized as a 

pathogen that will persist in low moisture foods for extended periods of time (30). 

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6. Is this product meant for higher risk population? 

In most instances, the product is made for consumption by the general population. 

7. What is the shelf life of the product? 

Months to years 

8. Would consumer handling and use be likely to increase or decrease risk? 

a. Heating for palatability 

b. Holding a frozen food under refrigeration 

c. Holding a refrigerated food beyond the use-by date 

If a thermal process was applied by the consumer for palatability, then the inherent pathogen risk 

relative to vegetative pathogens present due to contamination after processing might be mitigated to 

some extent depending on the process (time/temperature), but the heating might not fully eliminate the 

pathogen, depending on the number present. 

Example 1: Roasted and salted inshell pistachios 

Example 2: PPO-treated almond kernels 

Criteria a facility can apply to determine whether and how often to test ready-to-eat nuts processed for 

lethality: 

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299 Table D-3. Ready-to-eat nuts processed for lethality - Examples. 

Criterion/Factor Example 1: Roasted and salted inshell 

pistachios 

Ingredients: pistachios, sea salt 

Example 2: PPO-treated almond 

kernels 

Ingredients: almonds 

Process Information Raw pistachios are purchased directly 

from growers (suppliers) that may or 

may not be adhering to the produce 

safety regulation (21 CFR part 112). 

The roasting step is a process 

preventive control to significantly 

minimize Salmonella. Sanitation 

controls are implemented for the 

environment because the pistachios 

are exposed to the environment after 

roasting. 

Packed almonds are purchased from 

a supplier that treats the almonds 

with propylene oxide, a process 

control to significantly minimize 

Salmonella. The supplier does not 

expose the almonds to the 

environment after treatment. The 

receiving facility packages the 

almonds; because this facility relies 

upon its supplier to significantly 

minimize Salmonella in the almonds, 

they have established and 

implemented a supply-chain 

program. As the almonds are 

exposed to the environment after 

receiving and prior to packaging, 

they have established and 

implemented sanitation controls for 

the environment. 

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Criterion/Factor Example 1: Roasted and salted inshell 

pistachios 

Ingredients: pistachios, sea salt 

Example 2: PPO-treated almond 

kernels 

Ingredients: almonds 

A. Are pathogens 

associated with the food 

or ingredients? 

Yes – roasted, inshell pistachios have 

been linked to an outbreak of 

salmonellosis (34). 

Yes – raw almonds have been linked 

to outbreaks of salmonellosis (18, 

23). 

B. Are the ingredients likely Yes – a 2010-2012 survey of raw, Yes – surveys of raw almond kernels 

to be contaminated? inshell pistachios from storage silos 

found a Salmonella prevalence of 

0.81% (32 positive of 3,968 samples) 

(17) 

at processor receiving found a 

Salmonella prevalence of 0.98% 

(146 positive of 14,949 samples) 

(27). 

C. Are there robust Yes – roasting is expected to be a kill Yes – PPO treatment is expected to 

processing control step that would be established as a be a kill step that would be 

procedures such as a kill process preventive control to established as a process preventive 

step or other reduction significantly minimize pathogens such control to significantly minimize 

methods/controls? as Salmonella. pathogens such as Salmonella. 

D.Is there a potential for 

recontamination from the 

handling or the 

environment? 

Yes – roasted and salted pistachios are 

exposed to the production 

environment after roasting and prior 

to packaging. 

Yes, PPO treated almonds are 

exposed to the production 

environment after treatment and 

prior to packaging. 

E. Does the product support 

survival or growth? 

Salmonella will survive for extended 

periods of time in low moisture foods, 

including pistachios (26). 

Salmonella will survive for extended 

periods of time in low moisture 

foods, including almonds (26). 

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Criterion/Factor Example 1: Roasted and salted inshell 

pistachios 

Ingredients: pistachios, sea salt 

Example 2: PPO-treated almond 

kernels 

Ingredients: almonds 

F. Is this product meant for 

higher risk population? 

In most instances the product is being 

made for the general population 

In most instances the product is 

being made for the general 

population 

G.What is the shelf life of 

the product? 

Months to years Months to years 

H.Will consumer handling 

and use increase or 

decrease likelihood of 

pathogen survival, growth, 

or toxin production and 

risk of consumer illness? 

Risk may be increased due to 

consumer handling of the product as 

they remove shells, transferring 

contaminants (if present) from the 

shell to the nut kernel prior to 

consumption. Pathogen risk is reduced 

if cooked or baked. 

Pathogen risk is reduced if cooked 

or baked. 

300 

301 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

302 enzymes, would be an appropriate verification activity? 

303 No. 

304 

305 Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

306 evidence that the appropriate treatment was, in fact, applied? 

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Generally, finished product verification testing should be performed for most products in this 

category, even if there is evidence that the appropriate treatment was applied. Quantitative assessments 

of the risk of human salmonellosis from the consumption of almonds (32), pecans (32), pistachios (15), 

walnuts (31) (34), and peanuts (6) estimate the number of salmonellosis cases per year from the 

consumption of these nuts in the in the United States after various treatments (log reductions). These risk 

assessments include Salmonella dose-response models and U.S. consumption data. These risk 

assessments are appropriate sources of information facilities can use to establish target log reductions as 

part of the validation of their process controls, as required by subpart C of part 117; however, some 

atypical situations can occur that may result in pathogen prevalence and levels different from those in the 

dose-response models. Because of this, finished product testing as a verification of the effectiveness of a 

food safety plan and the facility’s capability to consistently deliver against it should be conducted. 

In addition to the finished product testing, a robust environmental monitoring program as a 

verification activity for sanitation preventive controls should be established, particularly if the treated 

nuts are exposed to these environments subsequent to their treatment. If there is a robust environmental 

monitoring program, verification testing of finished product could be less frequent. In-process indicator 

testing for hygiene monitoring, sanitation verification and evaluation of buildup on lines is necessary. 

Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

specific indicator organism) and type of test (e.g., presence/absence or enumeration)? What are 

appropriate indicator microorganisms for verifying processes adequately control pathogens? 

For processes that are not enclosed and for products that include post-lethality handling, finished 

product verification should be part of the preventive controls program. Selection of target organisms 

should be based on prevalence studies (cited above) and recall/outbreak information e.g., Salmonella and 

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L. monocytogenes (cited above). Quantitative indicator assays (Enterobacteriaceae, and/or coliforms) 

upon startup and in-process for process control (buildup of biofilms, water ingress, growth points) and 

sanitation verification may be used. 

Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

Base frequency of finished product testing on data from continuous programs that assess: 

1. Process control - develop an indicator/pathogen baseline to demonstrate process control for 

line/product. Microbiological testing should be performed with GMPs and processes in control to 

determine what is achievable and the variability that is normal. 

2. Environmental control - conduct extensive environmental monitoring to assess where and how often 

pathogens are found and detect harborage sites. 

3. Hazard assessment of ingredients, e.g., epidemiological information and prevalence of target 

pathogens in ingredients. 

4. Efficacy of sanitation programs, including studies conducted to determine frequency of sanitation and 

length of runs between cleaning and sanitizing activities. 

5. Level of hygiene segregation. 

6. Ability to determine in control/out of control quickly and see change when it occurs. 

Finished product testing may not be warranted (or may be limited) for validated processes verified to 

be under control; however, it is incumbent upon the manufacturer of treated nuts to determine if finished 

product testing for pathogens or indicator organisms would provide information useful to assess process 

control in their facility. Factors to consider when deciding to conduct finished product testing include who 

is doing the treatment, how rigorous is the treatment, process validation information, confidence in the 

entity doing the treatment, historical information from the supplier, how much exposure to the 

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environment and other factors. It is not uncommon for treated nut manufacturers to conduct finished 

product testing and supply a certificate of analysis with the products they ship. 

When finished product is screened for pathogens and/or coliforms or Enterobacteriaceae, testing a 

representative sample from a complete process run provides the most information about process 

control. A sampling plan and evaluation criteria should be developed based on the type of processed nut, 

the history of foodborne illness outbreaks associated with the processed nut, the storage and distribution 

chain, and the risk for cross contamination from the process environment. The sampling plan should 

clearly define what is considered the “lot”. FDA guidance provides an example of a sampling plan (sample 

size and number) when testing for Salmonella in foods that would not normally be subjected to a process 

lethal to Salmonella between the time of sampling and consumption (37). The sampling parameters are 

30 analytical units/ 25-gram samples. The samples may be aggregated into 375-gram analytical units. 

ICMSF provides additional information about finished product sampling plans commensurate with risk 

(22). 

Question 6: Are there situations in which testing at sites other than the end of the process can achieve the 

goal of verifying the adequacy of control of microbial hazards? 

No, for processes that are not enclosed, finished product testing is recommended, although 

additional points of verification testing are also important including: 

• Environmental monitoring –particularly in environments to which nuts are exposed after processing 

to reduce pathogens. 

• Inbound ingredient testing – depends on processed state of ingredients. If a manufacturer is relying 

on a supplier to control the hazard (e.g., a supplier of PPO-treats almonds to be packaged by the 

manufacturer), the incoming nuts may be subjected to testing to verify the supplier’s controls. If a 

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manufacturer is implementing a process control to significantly minimize the hazard (e.g., a 

manufacturer that roasts pistachios), the incoming nuts may not be subjected to testing. 

• Sanitation/hygiene verification testing. 

• Additional points of verification will not reduce the need for some level of finished product testing. 

• In addition, we would expect that for those products subjected to a thermal process, there would be 

a record of the process meeting critical limits, as well as the appropriate process validation. 

Question 7: What impact does environmental monitoring have on frequency and extent of product testing 

verification activities by companies? 

A robust environmental monitoring program (EMP) should be present or deployed targeting the 

post-lethality areas. Application of an EMP, however does not replace an active finished product 

verification testing program. 

Question 8: What criteria should a company apply in determining that microbial testing results indicate a 

loss of (systemic) process control? What actions should a company take if test results indicate a loss of 

control? When verification testing indicates loss of process control, to what extent should verification 

testing be increased, how far upstream and downstream should it go, and when and how should it be 

scaled back? 

Process control is based on delivery of a pathogen reduction treatment, either at the facility itself 

or by another entity. Control of the overall process is also based in part on effective sanitation as 

evidenced by environmental monitoring and finished product testing. Additionally, sanitation preventive 

controls and, in some cases, supply-chain preventive controls, and verification activities for these will 

contribute to the safety of the finished product. Note: Testing is predicated on established baselines for 

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401 the process and environment. This will require more frequent / intensive testing early on to establish a 

402 base line to demonstrate process control for this product / line. 

403 The finding of a pathogen in finished product is an indication of loss of process control. In addition, 

404 finding levels of indicator organisms above an established limit could indicate loss of control (or a trend 

405 toward loss of control). The types of actions that companies may take in the case of loss of control 

406 indicated by finished product verification results depend on the seriousness of the risk include the 

407 following: 

408 • Verification of process delivery (where a lethality process is delivered at the facility) 

409 • Verification of process delivery by a supplier (where the supplier applies the process) 

410 • Verification of the sanitation program 

411 • Increase verification testing frequency 

412 • Stop processing line until a root analysis is completed (unless running the process line is needed 

413 to help identify root cause) 

414 • Investigate the source of contamination under reduced production 

415 

416 Table D-4. Microbiological limits for RTE nuts processed for lethality. 

Target 

Microorganism 

Microbiological 

Limit 

Recommended Action if 

Limit is Exceeded 

Comments 

Salmonella Negative in two 

375 g samples 

Divert for reprocessing, if 

appropriate, or reject. Investigate 

and implement corrective action 

Two 375 g analytical units 

derived from 30 25 g samples 

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Listeria 

monocytogenes Negative in 25 g 

Reject lot, conduct root cause 

analysis and implement 

corrective action 

Investigative testing as 

response to EMP that suggests 

likely contamination of 

product 

Coliforms or 

Enterobacteriaceae 

<100 CFU/g 

Investigate, implement corrective 

action 

Root cause analysis is 

recommended 

417 Adapted from (29) 

418 

419 3. Ready-to-eat nut and seed products processed for lethality. 

420 The largest group in this category are the nut beverages (almond “milk”, macadamia “milk,” 

421 coconut “milk,” cashew “milk”, etc.). All of these products are commercially sold as pasteurized. The 

422 second major group includes multiple nut and seed spreads, cheese-like and yogurt-like products. 

423 

424 Question 1. What principles and criteria should a company apply in determining the need for and in 

425 designing an effective microbial testing program to verify that processes are effectively controlling 

426 microbial pathogens? 

427 1. Are pathogens associated with the food or ingredients? 

428 Given the diversity of products in this group and the multiple ingredients that they may contain, 

429 often added post-processing (such as in the case of spreads and yoghurts), it is quite likely that pathogens 

430 may be associated. There is one documented outbreak case of cashew cheese, but it had not been 

431 subjected to a lethality step (CDC, 2014). To this date there has not been foodborne illness caused by 

432 pathogenic organisms associated with nut beverages. 

433 

434 2. Are the ingredients likely to be contaminated? 

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The ingredients may be contaminated but once the liquid “milk” mixes are pasteurized and 

handled aseptically, all vegetative pathogens are eliminated. Similarly, other nut and seed products may 

be free from pathogen contamination after pasteurization of the nut beverage used for fermentation, but 

depending on post-processing handling, they may be subject to recontamination. 

3. Are there robust processing control procedures such as a kill step or other reduction methods/controls? 

Yes. Formulated nut products are derived from processed nuts and incorporate a pasteurization 

step such as HTST and UHT for nut beverages or other products. Nut “milks” are often subjected to aseptic 

packaging, so most contamination is controlled. HTST pasteurized nut “milks” still require refrigeration 

for preservation. The liquid components of other nut products may be pasteurized before fermentation 

and cheese making steps, but because of the post-lethality processing steps that expose products to the 

environment, refrigeration is critical to inhibit microbial growth. 

4. Is there a potential for inherent contamination or recontamination after processing from handling or 

the environment? 

The recontamination of nut “milks” is relatively unlikely given the use of HTST and UHT 

technologies with aseptic packaging. In the case of other nut and seed products, there is the possibility 

that they may be contaminated during the post-lethality steps with environmental contaminants such as 

Listeria monocytogenes, Salmonella and Staphylococcus aureus. 

5. Does the product support survival or growth? 

Nut “milks” processed with HTST are not commercially sterile and spoilage will occur in unopened 

packages. UHT nut milks are considered shelf-stable, and it is very unlikely that un-opened packages will 

experience spoilage. If the product is recontaminated after opening, nut “milks” can support the growth 

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of a wide variety of microorganisms. Microbial growth and survival in other nut products are highly likely, 

depending on pH and refrigeration temperatures. 

6. Is this product meant for higher risk population? 

In most instances all the products in this category are intended for the general population. 

7. What is the shelf life of the product? 

UHT-treated nut “milks” have 8 to 10 months of shelf life in unopened packages. Refrigerated 

HTST nut “milks” have shelf lives between 2 to 3 months in unopened packages. Most commercial brands 

recommend no more than 10 days for consumption after opening the product. 

The shelf life of other nut products is variable, depending on the intrinsic characteristics of the 

food product and ingredients. Fermented products with reduced water activity may have longer shelf 

lives. Additional ingredients such as nuts or spices may reduce the shelf life due to possible 

recontamination. 

8. Would consumer handling and use be likely to increase or decrease risk? 

Consumer handling will likely increase risk of any of the products in this category. Once nut 

beverages are opened, their shelf life decreases because of the potential for recontamination and the 

high susceptibility for microbial growth. Any of the products in this category may be subject to 

consumption beyond their expiration date. Cheese and yogurt-like products may be subjected to 

temperature abuse, which may increase their risk. 

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481 Example 1: Almond “milk” 

482 Example 2: Cashew Cheddar cheese 

483 

484 Table D-5. Ready-to-eat nut products processed for lethality – Examples. 

Criterion/Factor Example 1: Almond “milk” Example 2: Cashew Cheddar cheese 

Ingredients Ingredients: water, almonds, natural 

sweetener (sugar, cane syrup), salt, 

flavors (vanilla extract, other 

flavors), gelling agents (carrageenan, 

guar gum, gellan gum, xanthan gum, 

locust bean gum, starch), calcium 

salts (calcium carbonate, tri-calcium 

phosphate), sodium citrate, lecithin, 

vitamins (A, D2, E). 

Ingredients: cashew base (water, 

cashews), coconut oil, modified food 

starch (modified potato starch, 

modified cornstarch), potato starch, 

salt, natural flavors, dried yeast, 

vitamins (B1, B3, B6, B12), folic acid, 

annatto extract (for color), lactic 

acid, yeast extract, cultures. 

Process Information Almonds are grown in orchards, 

mechanically harvested by shaking 

the trees and allowed to dry on the 

floor for a few days. The nuts are 

harvested mechanically from the 

orchard floor. Nuts are cleaned, 

sorted and fumigated in windrows. 

Almonds are then graded, shelled 

and separated from shells. Shelled 

Cashew nuts are grown in evergreen 

tropical trees and almost all of them 

are imported. Cashews are harvested 

by manually separating them from 

the fruit. Inshell cashews are dried 

under sunlight for several days. Dry 

cashews are then sorted by size and 

treated with steam to loosen the 

shell. Shells are separated from nuts 

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Criterion/Factor Example 1: Almond “milk” Example 2: Cashew Cheddar cheese 

nuts are blanched, peeled and 

roasted. Roasted almonds are rinsed 

and soaked. Wet almonds are 

ground with water to obtain the 

almond “milk” paste. After this step, 

the “milk” paste is mixed with water 

and other ingredients, pasteurized 

using HTST or UHT processes and 

packaged aseptically. 

manually and re-dried in ovens at 

100 °C for 1 hour. Nuts are manually 

peeled, cleaned and sorted. Cashews 

are rinsed and soaked. Wet cashews 

are ground with water to obtain a 

cashew base. The cashew base is 

mixed with several ingredients and 

pasteurized. Starter cultures and 

gelling ingredients are added, and 

portions are molded. Cheese pieces 

are allowed to dry and mature. 

A. Are pathogens 

associated with the 

food or ingredients? 

Yes – raw almonds have been linked 

to outbreaks caused by Salmonella 

(7, 23) 

Yes, a cashew cheese was linked to a 

salmonellosis outbreak in 2014 (10). 

Salmonella has been detected in 

commercial cashew nuts (42). 

B. Are the ingredients Yes, pathogen contamination is Yes, pathogen contamination is 

likely to be considered reasonably likely for raw considered reasonably likely for raw 

contaminated? dry ingredients, in particular 

almonds. Salmonella prevalence has 

been reported to be 0.98% in raw 

almonds (27). 

dry ingredients, in particular 

cashews. 

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Criterion/Factor Example 1: Almond “milk” Example 2: Cashew Cheddar cheese 

C. Are there robust 

processing control 

procedures such as a 

kill step or other 

reduction 

methods/controls? 

Yes, almond “milk” is subjected to 

very effective thermal processes that 

eliminate pathogenic 

microorganisms. UHT and HTST 

processing are typically subjected to 

strict process controls. 

Cashew liquid base may be subjected 

to thermal treatment in order to 

fully cook starches that will serve as 

a kill step for controlling pathogens 

in the raw materials. In addition, the 

cheese mix is acidified and 

fermented with starter lactic acid 

cultures. 

D.Is there a potential for No, recontamination potential is Yes, handling of the product postrecontamination from extremely low because of immediate treatment may pose some risk of 

the handling or the post-lethality packaging under environmental recontamination 

environment? aseptic conditions. 

E. Does the product 

support survival or 

growth? 

Almond “milk” processes deliver 

commercially sterile products (UHT) 

or with extremely low counts (HTST). 

HTST “milks” may support growth, 

especially if they are not 

refrigerated. 

If the pH is maintained below 4.6, it 

will inhibit most pathogen growth, 

but spoilage by yeasts and molds can 

still occur. 

F. Is this product meant 

for higher risk 

population? 

In most instances the product is 

being made for the general 

population. 

The product is intended for the 

general population. 

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Criterion/Factor Example 1: Almond “milk” Example 2: Cashew Cheddar cheese 

G.What is the shelf life of 

the product? 

UHT almond “milk” – 8 to 10 

months. 

Refrigerated HTST almond “milk” - 2 

to 3 months. 

Depending on the handling, it could 

be up to 6 months. 

H.Will consumer 

handling and use 

increase or decrease 

likelihood of pathogen 

survival, growth, or 

toxin production and 

risk of consumer 

illness? 

Because the product is typically free 

from pathogens, it is unlikely that 

consumer handling would lead to 

pathogen recontamination. 

Temperature abuse may lead to 

growth of spoilage organisms 

previously present (HTST) or reintroduced with each serving. 

Consumer handling will increase the 

likelihood of recontamination, 

survival, and growth of pathogenic 

microorganisms 

485 

486 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

487 enzymes, would be an appropriate verification activity? 

488 There is no existing methodology that replaces pathogen and indicator microorganism testing. 

489 

490 Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

491 evidence that the appropriate treatment was, in fact, applied? 

492 Yes - if an enclosed pasteurization and filling process is used with the appropriate validation, 

493 verification testing may not be necessary. Batch processes are also used that would not be enclosed. In 

494 this case, verification testing would be needed. If there is a kill step, it would be designated a Process 

495 Preventive Control and a scientific validation study would be required. 

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Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

specific indicator organism) and type of test (e.g., presence/absence or enumeration)? What are 

appropriate indicator microorganisms for verifying processes adequately control pathogens? 

For processes that are not enclosed and for products that include post-lethality handling, finished 

product verification testing should be part of the preventive controls program. The selection of target 

organisms should be based on prevalence studies and recall/outbreak information e.g., Salmonella and L. 

monocytogenes. Quantitative indicator assays (Enterobacteriaceae, and/or coliforms) upon startup and 

in-process for process control (buildup of biofilms, water ingress, growth points) and sanitation 

verification may be used. 

Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

For products that use a non-enclosed system and for which production involves post-lethality 

handling, the following criteria are recommended: 

1. Base frequency of finished product testing on data from continuous programs that assess: 

• Process control - develop an indicator/pathogen baseline to demonstrate process control for 

line/product. 

• Environmental control - conduct extensive environmental monitoring to assess where and how 

often pathogens are found and detect harborage sites. 

2. Hazard assessment of ingredients - epidemiological and historic prevalence of target pathogens in 

ingredients. 

35 
 

 

 

 

 

 

 

 

 

 

 

 

 

519

520

521

522

523

524

525

526

527

528

529

530

531

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533

534

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536

537

538

539

540

541

542

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3. Efficacy of sanitation programs determined by including studies to determine frequency of sanitation 

and length of runs. 

4. Level of hygiene segregation. 

5. Ability to determine in control/out of control quickly and see change when it occurs. 

For beverage products manufactured under enclosed systems, criteria 2, 3, 4 and 5 should be 

considered. 

Question 6: Are there situations in which testing at sites other than the end of the process can achieve the 

goal of verifying the adequacy of control of microbial hazards? 

No, for processes that are not enclosed, finished product testing is recommended, although additional 

points of verification testing are also important including: 

• Environmental monitoring 

• Inbound raw material testing – depends on processed state of ingredients and COA data. Lot by 

lot testing may be needed if the supplier is deficient in pathogen mitigation interventions and 

hazards are not controlled by a process. 

• Sanitation/hygiene verification testing. 

Additional points of verification will not reduce the need for some level of finished product testing. 

In addition, we would expect that for those products subjected to a thermal process, there would be a 

record of the process meeting critical limits as well as the appropriate process validation. 

For enclosed processes, using a validated microbial reduction process, periodic end-product 

testing could be useful for verification of process control, but monitoring of process delivery should 

minimize the need for finished product testing. Environmental monitoring would not be needed if product 

is not exposed to the environment. 

36 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

543

544

545

546

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548

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Question 7: What impact does environmental monitoring have on frequency and extent of product testing 

verification activities by companies? 

For processes that are not enclosed, such as those used for other nut and seed products, a robust 

environmental monitoring program should be present or deployed targeting the post-lethality areas. 

Application of EMP, however does not replace active finished product verification. 

For enclosed processes, such as those used for nut beverages, environmental monitoring would 

not be needed if product is not exposed to the environment. 

Question 8: What criteria should a company apply in determining that microbial testing results indicate a 

loss of (systemic) process control? What actions should a company take if test results indicate a loss of 

control? When verification testing indicates loss of process control, to what extent should verification 

testing be increased, how far upstream and downstream should it go, and when and how should it be 

scaled back? 

To determine a loss of control, companies ideally should have in place several food safety plan 

components that include internal microbiological criteria, data recording, data analysis, and process 

control studies or charts. The process control charts should incorporate the previously set microbiological 

criteria and baseline data that would involve operating limits (upper and lower limits) that may result in 

actions in response to results. Depending on the type of microbial data (quantitative or qualitative) and 

criteria, control charts determine the times where the microbial testing results indicate that the results 

are beyond the operating limits. Based on the hazard analysis part of the food safety plan, the level of risk 

of the off-limits results will determine the tolerance level and the extent of out of compliance that would 

lead to a determination of a loss of process control. The control charts will detect consistent out of 

operating limits or trends within limits that would indicate loss of control. For example, for pathogen 

analysis results, a single positive result may trigger a corrective action that mandates the complete 

37 
 

 

 

 

 

 

 

 

 

 

 

 

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567 stoppage of the process. Indicator microorganism analyses may have a tolerance level for the number of 

568 out of operating limit results before a corrective action is taken. 

569 The types of actions that companies may take in the case of loss of control indicated by finished 

570 product verification results, depending on the seriousness of the risk, involve: 

571 1. Verification that process delivery was adequate 

572 2. Verification of sanitation program 

573 3. Increase verification testing frequency 

574 4. Stop processing line until a root analysis is completed 

575 5. Investigate the source of contamination under reduced production 

576 

577 Table D-6. Microbiological limits for nut and seed milks, cheeses and yogurts – RTE. 

Target Microorganism Microbiological 

Limit 

Recommended Action if 

Limit is Exceeded 

Comment 

Coliforms or 

Enterobacteriaceae 

< 10 CFU/g 

(milks) 

<100 CFU/g 

Investigate, implement 

corrective action 

Root cause analysis is 

recommended 

Listeria monocytogenes Negative in 25 g 

Reject lot, conduct root 

cause analysis and 

implement corrective 

action 

Investigative testing as 

response to EMP that 

suggests likely contamination 

of product 

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Reject lot, conduct root Two 375-g analytical units 

Salmonella Negative in 375 g 

cause analysis and 

implement corrective 

derived from 30 x 25-g 

samples 

action 

578 

579 4. Ready-to-eat nut/seed butters not processed for lethality beyond initial nut/seed processing 

580 This group of RTE products includes a variety of butters in which the main ingredient is a nut type 

581 and the level of moisture is relatively low. Commercially available butters are made of almonds, cashews, 

582 hazelnut, peanuts, pistachios, sesame seeds, sunflower seeds, walnuts, etc. In most cases, the whole nuts 

583 have been subjected to a thermal treatment such as roasting, which should have been validated. After 

584 roasting, butters are produced by grinding the nuts to obtain a paste that can be spread. Depending on 

585 the extent of grinding, their texture can be smoother. Other ingredients such as sugar, vegetable oils, and 

586 salt are often added. 

587 

588 Question 1. What principles and criteria should a company apply in determining the need for and in 

589 designing an effective microbial testing program to verify that processes are effectively controlling 

590 microbial pathogens? 

591 1. Are pathogens associated with the food or ingredients? 

592 Yes, nut and seed butters have been linked to outbreaks from Salmonella and E. coli O157:H7 (8, 

593 9, 12). 

594 2. Are the ingredients likely to be contaminated? 

595 Yes, raw nuts and seeds are likely to be contaminated; there are documented outbreaks and 

596 recalls linked to multiple nut types that may lead to contaminated butters. 

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597

598

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3. Are there robust processing control procedures such as a kill step or other reduction 

methods/controls? 

Yes, some manufacturers may apply several forms of processing to significantly minimize or 

prevent biological hazards in nuts and seeds before they are ground. Such processes would likely be 

process controls and include oil roasting, dry roasting, toasting, propylene oxide treatment, steam 

treatment, and blanching. 

4. Is there a potential for inherent contamination or recontamination after processing from 

handling or the environment? 

Yes, nut and seed butters may be inherently contaminated if the raw nuts or seeds had not been 

subjected to one of the processes described above and they may be susceptible to recontamination during 

mixing, grinding and packaging. 

5. Does the product support survival or growth? 

Salmonella, E. coli O157 and Listeria monocytogenes are capable of surviving in nut and seed 

butters for very long periods of time during storage, but since most nut/seed butters have relatively low 

water activity their growth is markedly inhibited(3, 24, 25). Typically, none of the nut butters require 

refrigeration for storage because of their low water activity, although manufacturers may recommend 

refrigeration after opening to retard rancidity. 

6. Is this product meant for higher risk population? 

The product is intended for the general population. 

7. What is the shelf life of the product? 

Most nut and seed butters will have shelf-life periods 6 to 12 months. Their shelf life will be limited 

by how susceptible their fat is to rancidity; microbial growth is not a factor for shelf-life determination. 

8. Would consumer handling and use be likely to increase or decrease risk? 

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620 The risk of shelf-stable nut and seed butters may not be affected by consumer handling because 

621 of their low water activity. Temperature abuse will not lead to microbial growth. Pathogen levels should 

622 slowly decline during ambient temperature storage. 

623 

624 Table D-7. Ready-to-eat nut/seed butters not processed for lethality beyond initial nut/seed 

625 processing – Examples. 

Criterion/Factor Example 1: Peanut butter Example 2: Tahini 

Ingredients Ingredients: dry roasted peanuts, 

sugar, vegetable oil (hydrogenated 

or palm oil), salt, molasses 

Ingredients: sesame seeds 

Process Information Peanuts are grown from a legume 

plant as row crops. Peanuts are 

mechanically harvested from the 

soil, allowed to dry in the field for 2 

to 3 days, separated from the vine 

while being picked from the floor. 

Inshell peanuts will be heat dried to 

reach 8-10% moisture. Peanuts will 

be graded, shelled, and sorted. 

Peanuts are then roasted at more 

than 150°C, cooled, and dry 

blanched to remove the skins. The 

Sesame seeds are grown in plants of 

the Pedaliaceae family. Mature 

sesame plants are harvested, 

bundled, and allowed to dry on soil. 

Seeds are collected and separated 

from the sickles when moisture level 

is less than 8%. Seeds are washed, 

dried by different methods, and 

roasted. Roasted seeds are cooled 

then milled to form a paste. Sesame 

paste is degassed and packaged. 

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Criterion/Factor Example 1: Peanut butter Example 2: Tahini 

nuts are ground, mixed with other 

ingredients, and packaged. 

A. Are pathogens 

associated with the 

food or ingredients? 

Yes, there have been several 

salmonellosis outbreaks linked to 

peanut butter (CDC, 2007, 2009). 

Yes, there have been several 

outbreaks caused by Salmonella 

linked to tahini (13, 35). 

B. Are the ingredients Several surveys of raw shelled The prevalence of Salmonella in 

likely to be peanuts have reported Salmonella imported sesame seeds was 

contaminated? prevalence from 0.14 to 1.6% (4, 28). 

If the blanching or roasting process is 

not validated to provide adequate 

microbial reduction, peanuts used in 

processing could be contaminated. 

determined to be almost 10% by the 

FDA (40). If non-roasted seeds are 

used or if the roasting process is not 

validated to provide adequate 

microbial reduction, sesame seeds 

are likely to be contaminated with 

Salmonella. 

C. Are there robust 

processing control 

procedures such as a 

kill step or other 

reduction 

methods/controls? 

A validated blanching, roasting, or 

other kill step may be used. 

A validated roasting or other kill step 

may be used. 

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Criterion/Factor Example 1: Peanut butter Example 2: Tahini 

D.Is there a potential for 

recontamination from 

the handling or the 

environment? 

Yes, during grinding and packing the 

product can be recontaminated from 

equipment. 

Yes, during grinding and packing the 

product can be recontaminated from 

equipment. 

E. Does the product 

support survival or 

growth? 

Its low water activity inhibits most 

microorganisms’ growth, but 

pathogens such as Salmonella may 

survive for several months. 

Tahini’s low water activity inhibits all 

microbial growth, but Salmonella is 

capable of surviving for a long time. 

F. Is this product meant 

for higher risk 

population? 

While this product is intended for 

the general population, children are 

probably the largest segment that 

consumes peanut butter. 

This product is intended for the 

general population. 

G.What is the shelf life of 

the product? 

Most commercial brands have 1-year 

shelf-life expectations 

The shelf life of commercial products 

ranges from 1 to 2 years. 

H.Will consumer 

handling and use 

increase or decrease 

likelihood of pathogen 

survival, growth, or 

toxin production and 

risk of consumer 

illness? 

No, because of the low water 

activity, consumer handling would 

not influence survival or growth. 

No, because of the low water 

activity, consumer handling would 

not influence survival or growth. 

43 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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631

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643

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Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

enzymes, would be an appropriate verification activity? 

There are no existing assays/testing that replaces pathogen and indicator microorganism 

testing. 

Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

evidence that the appropriate treatment was, in fact, applied? 

No. Finished product testing in addition to a robust environmental monitoring program (primarily 

Zones 2 and 3) is recommended. In-process indicator testing for hygiene monitoring, sanitation 

verification, and evaluation of buildup on lines is necessary. 

Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

specific indicator organism) and type of test (e.g., presence/absence or enumeration)? What are 

appropriate indicator microorganisms for verifying processes adequately control pathogens? 

Finished product should be screened. Microbiological limits should be set for target 

microorganisms selected based on prevalence studies and recall/outbreak information e.g., Salmonella 

and L. monocytogenes. Quantitative indicator assays (Enterobacteriaceae and/or coliforms) should be 

conducted upon startup and in-process for process control (buildup of biofilms, water ingress, growth 

points) and sanitation verification. 

Question 5. What principles and criteria should a company apply in determining the frequency of testing 

finished product to determine if the company’s food safety system for that product is effective? 

44 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

650

651

652

653

654

655

656

657

658

659

660

661

662

663

664

665

666

667

668

669

670

671

672

673

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1. Base frequency of finished product testing on data from continuous programs that assess: 

• Process control - develop an indicator/pathogen baseline to demonstrate process control for 

line/product. 

• Environmental control – conduct extensive environmental testing to assess where and how often 

pathogens are found and detect harborage sites. 

2. Hazard assessment of ingredients: Prevalence of target pathogens in ingredients. 

3. Efficacy of sanitation programs - including studies to determine frequency of sanitation and length of 

runs. 

4. Level of hygiene segregation. 

5. Ability to determine in control/out of control quickly and see change when it occurs. 

Question 6: Are there situations in which testing at sites other than the end of the process can achieve the 

goal of verifying the adequacy of control of microbial hazards? 

No. Finished product testing is needed, although additional points of verification testing are also 

important including: 

• Environmental monitoring 

• Inbound raw material testing for processed nuts sourced externally if a risk-based supplier 

program is not in place. A good supplier program involves regular audits to confirm the presence 

of a validated process, sanitation controls and a robust environmental monitoring program. Raw 

nuts sourced for internal roasting would not be tested, since the control is being applied in-house; 

hygiene segregation controls should be in place. Sanitation/hygiene verification testing should be 

conducted. 

Additional points of verification will not negate need for some level of finished product testing. 

45 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

674

675

676

677

678

679

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687

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689

690

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693

694

695

696

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Question 7: What impact does environmental monitoring have on frequency and extent of product testing 

verification activities by companies? 

Environmental monitoring should be an integral component of the testing program of companies 

manufacturing nut and seed butters. The deployment of environmental monitoring complements the 

application of finished product verification testing and along with implementation of effective process 

controls can reduce the amount of finished product testing needed. 

Question 8: What criteria should a company apply in determining that microbial testing results indicate a 

loss of (systemic) process control? What actions should a company take if test results indicate a loss of 

control? When verification testing indicates loss of process control, to what extent should verification 

testing be increased, how far upstream and downstream should it go, and when and how should it be 

scaled back? 

To determine a loss of control, companies ideally should have in place several food safety plan 

components that include internal microbiological criteria, data recording, data analysis and process 

control studies or charts. The process control charts should incorporate the previously set microbiological 

criteria and baseline data that would involve operating limits (upper and lower limits) that may result in 

actions in response to results. Depending on the type of microbial data (quantitative or qualitative) and 

criteria, control charts determine the times where the microbial testing results indicate that the results 

are beyond the operating limits. Based on the hazard analysis of the food safety plan, the level of risk of 

the off-limits results will determine the tolerance level and the extent of out of compliance that would 

lead to determine a loss of process control. The control charts will detect consistent out of operating limits 

or trends within limits that would indicate loss of control. For example, for pathogen analysis results, a 

single positive result may trigger a corrective action that mandates the complete stoppage of the process. 

46 
 

 

 

 

 

 

 

 

 

 

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697 For indicator microorganisms, analyses may have a tolerance level of the number of out of operating limits 

698 results before a corrective action is taken. 

699 The types of actions that companies may take in the case of loss of control indicated by finished 

700 product verification results depending on the seriousness of the risk involve: 

701 1. Verification that process delivery was adequate 

702 2. Verification of sanitation program 

703 3. Increase verification testing frequency 

704 4. Stop processing line until a root analysis is completed 

705 5. Investigate the source of contamination under reduced production 

706 

707 Table D-8. Microbiological limits for nut and seed butters – RTE 

Target 

Microorganism 

Microbiological 

Limit 

Recommended Action if 

Limit is Exceeded 

Comments 

Coliforms or 

Enterobacteriaceae 

<100 CFU/g 

Investigate, implement 

corrective action 

Root cause analysis is 

recommended 

Listeria 

monocytogenes 

(product) 

Negative in 25 g 

Reject lot, conduct root 

cause analysis and 

implement corrective 

action 

Investigative testing as 

response to EMP that 

suggests likely 

contamination of product 

Salmonella 

(product) 

Negative in two 375 

g samples 

Reject. Investigate and 

implement corrective 

action 

Two 375g analytical units 

derived from 30 25 g 

samples 

708 Adapted from (29) 

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709

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711

712

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715

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719

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721

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723

724

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727

728

729

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731

732

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Microorganisms in Foods 7: Microbiological Testing in Food Safety Management. 2nd edition. Springer 

International Publishing, New York. 

23. Isaacs, S., J. Aramini, B. Ciebin, J. A. Farrar, R. Ahmed, D. Middleton, A. U. Chandran, L. J. Harris, 

M. Howes, E. Chan, A. S. Pichette, K. Campbell, A. Gupta, L. Y. Lior, M. Pearce, C. Clark, F. Rodgers, F. 

Jamieson, I. Brophy, and A. Ellis. 2005. An International outbreak of salmonellosis associated with raw 

almonds contaminated with a rare phage type of Salmonella Enteritidis. J. Food Prot. 68:191-198. 

24. Kataoka, A., E. Enache, D. G. Black, P. H. Elliott, C. D. Napier, R. Podolak, and M. M. Hayman. 2014. 

Survival of Salmonella Tennessee, Salmonella Typhimurium DT104, and Enterococcus faecium in peanut 

paste formulations at two different levels of water activity and fat. J. Food Prot. 77:1252-9. 

25. Kenney, S. J., and L. R. Beuchat. 2004. Survival, growth, and thermal resistance of Listeria 

monocytogenes in products containing peanut and chocolate. Journal of Food Protection. 67:2205-2211. 

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26. Kimber, M. A., H. Kaur, L. Wang, M. D. Danyluk, and L. J. Harris. 2012. Survival of Salmonella, 

Escherichia coli O157: H7, and Listeria monocytogenes on inoculated almonds and pistachios stored at−

19, 4, and 24° C. J. Food Prot. 75:1394-1403. 

27. Lambertini, E., M. D. Danyluk, D. W. Schaffner, C. K. Winter, and L. J. Harris. 2012. Risk of 

salmonellosis from consumption of almonds in the North American market. Food Res Int. 45:1166-1174. 

28. Miksch, R. R., J. Leek, S. Myoda, T. Nguyen, K. Tenney, V. Svidenko, K. Greeson, and M. Samadpour. 

2013. Prevalence and counts of Salmonella and enterohemorrhagic Escherichia coli in raw, shelled runner 

peanuts. J. Food Prot. 76:1668-1675. 

29. National Advisory Committee for Microbiological Criteria for Foods. 2018. Response to questions 

posed by the Department of Defense regarding microbiological criteria as indicators of process control or 

insanitary conditions. J. Food Prot. 81:115-141. 

30. Santillana Farakos, S. M., R. Pouillot, N. Anderson, R. Johnson, I. Son, and J. Van Doren. 2016. 

Modeling the survival kinetics of Salmonella in tree nuts for use in risk assessment. Int J Food Microbiol. 

227:41-50. 

31. Santillana Farakos, S. M., R. Pouillot, G. R. Davidson, R. Johnson, I. Son, N. Anderson, and J. M. Van 

Doren. 2019. A quantitative risk assessment of human salmonellosis from consumption of walnuts in the 

United States. J. Food Prot. 82:45-57. 

32. Santillana Farakos, S. M., R. Pouillot, R. Johnson, J. Spungen, I. Son, N. Anderson, and J. M. Van 

Doren. 2017. A quantitative assessment of the risk of human salmonellosis arising from the consumption 

of almonds in the United States: the impact of preventive treatment levels. J. Food Prot. 80:863-878. 

33. U. S. Food and Drug Administration. 2015. FSMA Final Rule for Preventive Controls for Human 

Food-Current Good Manufacturing Practice, Hazard Analysis, and Risk-Based Preventive Controls for 

Human Food; Final Rule. https://www.fda.gov/Food/GuidanceRegulation/FSMA/ucm334115.htm, 

Accessed April 14, 2021. 

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34. U. S. Food and Drug Administration. 2016. FDA investigated multistate outbreak of Salmonella 

infections linked to Wonderful Pistachio. https://www.fda.gov/food/outbreaks-foodborne-illness/fdainvestigated-multistate-outbreak-salmonella-infections-linked-wonderful-pistachio#:~:text=On, 

Accessed April 14, 2021. 

35. U. S. Food and Drug Administration. 2020. FDA Fact Sheet Produce Safety Rule (21 CFR 112) – 

Rarely Consumed Raw Produce. https://www.fda.gov/files/food/published/FDA-Fact-Sheet-ProduceSafety-Rule-21-CFR-112-Rarely-Consumed-Raw-Produce-PDF.pdf, Accessed April 14, 2021. 

36. U.S. Food and Drug Administration. 2005. CPG Sec 570.450 Tree Nuts - Adulteration with filth, 

involving the presence of the organism Escherichia coli. https://www.fda.gov/regulatoryinformation/search-fda-guidance-documents/cpg-sec-570450-tree-nuts-adulteration-filth-involvingpresence-organism-escherichia-coli, Accessed April 14, 20212. 

37. U.S. Food and Drug Administration. 2015. Investigations Operations Manual 2015, Ch. 4 Sampling. 

http://www.fda.gov/downloads/ICECI/Inspections/IOM/UCM123507.pdf, Accessed April 13, 2021. 

38. U.S. Food and Drug Administration. 2019. Guidance for Industry: Enforcement Policy for Entities 

Growing, Harvesting, Packing, or Holding Hops, Wine Grapes, Pulse Crops, and Almond. 

https://www.fda.gov/regulatory-information/search-fda-guidance-documents/guidance-industryenforcement-policy-entities-growing-harvesting-packing-or-holding-hops-wine-grapes, Accessed April 

14, 2021. 

39. U.S. Food and Drug Administration. 2021. 21 CFR Part 112—Standards for the growing, harvesting, 

packing, and holding of produce for human consumption. https://ecfr.federalregister.gov/current/title21/chapter-I/subchapter-B/part-112?toc=1, Accessed April 14, 2021. 

40. Van Doren, J. M., R. J. Blodgett, R. Pouillot, A. Westerman, D. Kleinmeier, G. C. Ziobro, Y. Q. Ma, 

T. S. Hammack, V. Gill, M. F. Muckenfuss, and L. Fabbri. 2013. Prevalence, level and distribution of 

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Salmonella in shipments of imported capsicum and sesame seed spice offered for entry to the United 

States: Observations and modeling results. Food Microbiology. 36:149-160. 

41. Yada, S., and L. J. Harris. 2019. Recalls of tree nuts and peanuts in the U.S., 2001 to present (version 

2) [Table and references]. In U.S. recalls of nuts. . http://ucfoodsafety.ucdavis.edu/Nuts_and_Nut_Pastes, 

Accessed April 14, 2021. 

42. Zhang, G., L. Hu, D. Melka, H. Wang, A. Laasri, E. W. Brown, E. Strain, M. Allard, V. K. Bunning, S. 

M. Musser, R. Johnson, S. M. S. Farakos, V. N. Scott, R. Pouillot, J. M. Van Doren, and T. S. Hammack. 2017. 

Prevalence of Salmonella in cashews, hazelnuts, macadamia nuts, pecans, pine nuts, and walnuts in the 

United States. J. Food Prot. 80:459-466. 

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1 APPENDIX E - CATEGORY: FRUITS AND VEGETABLES 

2 • RTE fresh-cut fruits (e.g., cut melon, sectioned grapefruit, sliced pineapple) 

3 • RTE fresh-cut vegetables, including packaged leafy greens (e.g., cut celery stalks, peeled baby 

4 carrots, sliced mushrooms, shredded cabbage, chopped lettuce, spring mix) 

5 • RTE dried/dehydrated fruits (e.g., dried cranberries, raisins, dried apricots) 

6 This category includes any fresh fruit or vegetable or combination that has been physically altered 

7 from its whole state after being harvested from the field (e.g., by chopping, dicing, peeling, ricing, 

8 shredding, slicing, spiralizing, or tearing) without additional processing (such as blanching or cooking). 

9 Fresh-cut produce may or may not undergo a wash or other treatment before being packaged for use by 

10 the consumer or a retail food establishment. Fresh-cut produce can be a single commodity or two or more 

11 mixed in the same package, such as garden salad kits, coleslaw, or fruit salads, and sometimes called 

12 “ready to use,” “precut,” or “value added” produce. Fresh-cut produce also does not include produce that 

13 has been processed by freezing, cooking, canning, or packing in a juice, syrup, or dressing. For the 

14 purposes of this document, only RTE fresh-cut fruits and vegetables to be consumed as such were 

15 considered; it does not apply to fresh-cut produce that require cooking (such as cut butternut squash). 

16 Leafy greens have been most frequently associated with outbreaks of shiga-toxin producing E. coli (7). 

17 The risk profile of leafy greens can be differentially linked to four categories 1) type of leafy greens; 2) 

18 growing locations; 3) harvesting practices and 4) processing practices. However, the pathogens 

19 associated with outbreaks (i.e., STEC) are widely acknowledged to have origins in very low levels of 

20 sporadic and diffuse contamination within the growing environment and during harvest. Investigative 

21 studies have yielded positive samples, primarily from water and sediments, in the implicated growing 

22 locations. These positive environmental samples have been recovered from water sources, including both 

23 irrigation canals and on-farm reservoirs generally near animal feeding operations or pasture-managed 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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24 animals. However, these may be more correlations of the environment than causative sources of the 

outbreak; significant knowledge gaps exist. Preventing contamination of leafy greens in the growing 

26 environment and subsequently prevention of its amplification during harvesting and processing is the key 

27 focus. In practical terms, testing can be applied in the growing environment as a monitoring tool for gross 

28 contamination, but direct pathogen testing of harvested commodity lettuces or bagged fresh cut leafy 

29 greens is not generally viewed as useful because of the extremely low level of contamination and the time 

it takes for to receive results (frequently minimum of 2-3 days on a 17-day shelf-life). Growers should 

31 consult the guidelines for production and harvest of leafy greens (12); postharvest processing should rely 

32 on tight hygienic controls of the wash water, especially recirculated water, to lower contaminant residuals 

33 on product and prevent cross-contamination (3). 

34 Note: It has been well established that most microorganisms grow best at pH values around 7.0 (6.0– 

7.5), whereas few grow below 4.0 (4). When reading this document please keep in mind that many fruits 

36 (e.g., citrus fruits) fall below the point at which bacteria normally grow because of their low pH (pH <4), 

37 whereas others (e.g., melons, bananas, and papaya) can support growth (6). In contrast, most vegetables 

38 have pH values near neutrality (or slightly lower e.g., cucumber, carrots), therefore they are more 

39 supportive of bacterial growth. 

1. RTE fresh-cut fruits 

41 Examples include melon, sectioned grapefruit, sliced pineapple. These items are usually consumed 

42 raw, without a treatment that adequately reduces pathogens (i.e., a “kill step”). However, fruits with low 

43 pH (below 4) can help prevent the growth of certain pathogens such as Listeria monocytogenes and 

44 Salmonella (5). However, fruits with high pH values, e.g., melon, bananas and papaya (5), can support 

growth; therefore an important aspect of applying the information in this document is to consider the pH 

46 of RTE fresh-cut fruit products. 

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48 2. RTE fresh-cut vegetables, including packaged leafy greens 

49 Examples include cut celery stalks, peeled baby carrots, sliced mushrooms, shredded cabbage and 

packaged leafy greens (e.g., chopped lettuce, spring mix). These items are usually consumed raw; the 

51 facility provides no microbial reduction step other than physical removal by cleaning, peeling, and 

52 washing. Most vegetables have pH values near neutrality; therefore, they are more subject to bacterial 

53 growth from pathogens such as Listeria monocytogenes, pathogenic Escherichia coli and Salmonella. 

54 Question 1. What principles and criteria should a company apply in determining the need for and in 

designing an effective microbial testing program to verify that processes are effectively controlling 

56 microbial pathogens? 

57 Principles that apply to finished product testing of fresh-cut fruits and vegetables: 

58 a. Microbiological testing is most useful (1) if ingredients in a food have the potential to contain 

59 pathogens and there is no kill step (such as the case of RTE fresh-cut fruits) in the manufacture of the 

finished product, and/or (2) when finished products may be contaminated from the environment. 

61 Since these products contains raw ingredients that have not received a kill step and they are also 

62 exposed to the environment during preparation and filling of containers, they could be contaminated 

63 with L. monocytogenes, Salmonella spp. and pathogenic Escherichia coli, but parasites and viruses 

64 from growing and harvest could also be present. 

b. Based on the above point, the outbreak history of the commodity plays a key role, as well as 

66 seasonality (especially for parasites), therefore looking at updated information on outbreaks from 

67 CDC (2) and FDA (10) is important in determining control measures to ensure product safety. 

68 c. Use of microbiological testing as verification of control measures should consider risk to the 

69 consumer. Immunocompromised, infants, pregnant women, and elderly are more susceptible 

consumers, depending on the pathogen. Low pH (<4.0) fresh-cut fruits naturally inhibit microbial 

71 growth. The risk to consumers is higher when pH of final product is above 4.0 or if a product with pH 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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72 >4.4 has been kept at refrigerated temperature for extended periods, which supports the growth of 

73 L. monocytogenes. 

74 d. Microbiological testing should be increased when data indicate that the operation is not under control 

75 (e.g., records indicate a deviation at a CCP, a pathogen has been detected on a food contact surface 

76 or in the finished product). 

77 Criteria a facility can apply to determine whether and how often to test ready-to-eat 

78 fresh-cut fruit and vegetable products: 

Criterion/Factor RTE fresh-cut fruits RTE fresh-cut vegetables 

A. Are pathogens 

associated with the 

food or ingredients? 

Yes, fresh-cut fruits have been 

associated with pathogens, including 

viruses (2). 

Yes, fresh-cut vegetables have been 

associated with pathogens, parasites 

and viruses (2) 

B. Are the ingredients 

likely to be 

contaminated? 

Yes, and supplier verification 

programs are necessary for some 

ingredients. Each ingredient needs 

to be assessed depending on 

commodity type and its attributes 

(e.g., pH, water content, rind/peel) 

Yes, and supplier verification 

programs are necessary for some 

ingredients. Each ingredient needs 

to be assessed depending on 

commodity type and its attributes 

(e.g., pH, water content, rind/peel) 

C. Are there robust 

processing control 

procedures such as a 

kill step or other 

Antimicrobials in produce washes 

are typically used to prevent cross 

contamination in the wash water 

and not as a microbial reduction step 

on the product surface. Suppliers of 

Antimicrobials in produce washes 

are typically used to prevent cross 

contamination in the wash water 

and not as a microbial reduction step 

on the product surface. Suppliers of 
 

 

 

 

 

 

 

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Criterion/Factor RTE fresh-cut fruits RTE fresh-cut vegetables 

reduction fruits and vegetables for fresh-cut or fruits and vegetables for fresh-cut or 

methods/controls? drying should comply with the 

Produce Safety Rule (21 CFR part 

112) where applicable, or GAPs. 

Some drying processes may have 

sufficient heat to inactivate 

pathogens. 

drying should comply with the 

Produce Safety Rule (21 CFR part 

112) where applicable, or GAPs. 

Some drying processes may have 

sufficient heat to inactivate 

pathogens. 

D. Is there a potential for Yes, the product is exposed to the Yes, the product is exposed to the 

recontamination from environment during ingredient environment during ingredient 

the handling or the preparation (e.g., chopping); preparation (e.g., chopping); 

environment? however, sanitation controls and a 

robust environmental monitoring 

program (EMP) can reduce the 

potential for contamination with 

microbial pathogens. 

however, sanitation controls and a 

robust EMP can reduce the potential 

for contamination with microbial 

pathogens. 

E. Does the product 

support survival or 

growth? 

Pathogens will survive on fresh cut 

fruits. 

Pathogens will survive on fresh cut 

vegetables 
 

 

 

 

 

 

 

 

 

 

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Criterion/Factor RTE fresh-cut fruits RTE fresh-cut vegetables 

F. Is this product meant 

for higher risk 

population? 

In most instances the product is 

being made for the general 

population. 

In most instances the product is 

being made for the general 

population. 

G. What is the shelf life of 

the product? 

1 week 1 week 

H. Will consumer handling 

and use increase or 

decrease risk of 

pathogen survival, 

growth, or toxin 

production? 

Yes, if improper handling and 

temperature abuse occurs, the risk 

of pathogen growth increases in 

fruits with a pH supporting pathogen 

growth, if pathogens are present. 

Yes, if improper handling and 

temperature abuse occurs. 

79 

80 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

81 enzymes, would be an appropriate verification activity? 

82 Yes, monitoring the wash system parameters (e.g., washing time, water temperature, 

83 antimicrobial concentration, organic load as identified for a CCP in a food safety plan) may be used as 

84 appropriate verification activities for the process preventive control for biological hazards in wash water. 

85 However, these would not be an appropriate alternative to pathogen or indicator testing to verify supplier 

86 controls or sanitation controls in the processing facility. 

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88 Question 3. Are there situations where [microbial] verification testing would not be necessary if there 

89 is evidence that the appropriate treatment was, in fact, applied? 

RTE fresh-cut fruits and vegetables are not subjected to a kill step. However, antimicrobials should 

91 be used in wash water as a process preventive control for biological hazards to prevent cross92 contamination during washing. Microbial testing of fresh-cut produce as a verification activity of this 

93 process control may not be necessary if other verification activities (e.g., a review of process logs for the 

94 sensor continuously monitoring the antimicrobial concentration in the wash water) indicate the process 

is being consistently implemented. However, pathogen or indicator testing may still be appropriate to 

96 verify supplier controls or sanitation controls in the processing facility. 

97 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

98 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

99 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

101 Considerations in selecting the test organism include outbreak and recall information for the 

102 ingredients or finished products, the lack of a process control to adequately reduce pathogens (i.e., a “kill 

103 step”), the types and adequacy of controls applied by suppliers (the growers), and the likelihood of 

104 recontamination in the production environment. Routine in-process or finished product testing for 

generic E. coli and environmental testing for Listeria (11) should be used to assess process control and 

106 insanitary conditions. Generic E. coli is a better indicator of process control and sanitation than pathogenic 

107 E. coli, because generic E. coli would be present in greater numbers. 

108 Question 5. What principles and criteria should a company apply in determining the frequency of testing 

109 finished product to determine if the company’s food safety system for that product is effective? 

Preventive controls for raw ingredients should be established and implemented through a supply111 chain program, as there is no kill step at any point in the production of fresh-cut fruits and vegetables. As 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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112 part of a supply-chain preventive controls program, annual onsite audits of each approved supplier (i.e., 

113 growers), including an assessment of the farm’s procedures, processes, and practices during growing, 

114 harvesting, packing, and holding, as related to compliance with the Produce Safety Rule or GAPs are 

needed (9). If the sanitation program, process controls for biological hazards (such as the use of 

116 antimicrobials in wash water to significantly minimize or prevent cross-contamination of pathogens), and 

117 the supply-chain program are all robust, the frequency of testing finished product may be less than when 

118 such programs are deficient. The frequency of such testing should be sufficient to demonstrate control. 

119 Testing frequency may increase (e.g., from weekly to daily) based on emerging issues (e.g., an on-going 

outbreak), EMP results, and seasonality considerations (particularly for parasites). 

121 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

122 the goal of verifying the adequacy of control of microbial hazards? 

123 NACMCF (8) indicated that periodic testing by suppliers of in-process or finished products for 

124 Salmonella or E. coli O157:H7 (or other appropriate STEC) may be pertinent depending on the commodity, 

geographic location, and use of GAPs for various commodities (see 

126 https://www.ams.usda.gov/services/auditing/gap-ghp or other FDA or university guidance documents 

127 and training). Due to a relatively short shelf-life of fresh-cut produce, finished product testing that may 

128 require a “test and hold” could be a limitation to regular end-product testing as a verification activity. 

129 Accordingly, reliance on testing and other verification activities at other sites may decrease the frequency 

of finished product testing. Such testing or other activities include the following: annual onsite audits of 

131 suppliers and preharvest testing as part of a supply-chain program; environmental monitoring as a 

132 verification of a sanitation program; and robust process controls and associated verification activities for 

133 biological hazards in wash water (i.e., use of antimicrobials to significantly minimize or prevent the cross134 contamination of biological hazards). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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136 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

137 product testing verification activities by companies? 

138 A robust EMP could reduce the amount of finished product testing, as some of the pathogens of 

139 concern (e.g., L. monocytogenes, Salmonella) can reside in the production environment and could result 

140 in environmental contamination. The source of these environmental pathogens is commonly raw 

141 produce; since pathogens associated with fresh-cut produces are likely to come in on the produce, 

142 product testing can be an important verification of control measures from farm to finished product, with 

143 environmental monitoring addressing the potential for contamination from the processing environment. 

144 There is an interrelatedness of the testing program with other controls such as supplier audits and process 

145 controls for wash water, along with GMPs in the plant, that should be used to determine the frequency 

146 and extent of finished produced verification testing. 

147 Question 8: What criteria should a company apply in determining that microbial testing results indicate 

148 a loss of (systemic) process control? 

149 What actions should a company take if test results indicate a loss of control? 

150 When verification testing indicates loss of process control, to what extent should verification testing be 

151 increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

152 If generic E. coli exceeds a defined limit (e.g., 100 CFU/g) investigation into the cause is warranted. 

153 In some cases, product testing for pathogens such as STEC, Salmonella and L. monocytogenes may be 

154 warranted. Corrective actions should be taken for any finding of L. monocytogenes in the environment; 

155 corrective actions followed by repeat positives may indicate the need of more frequent product testing 

156 for L. monocytogenes (see FDA draft guidance on Control of L. monocytogenes in RTE Foods (11)) until the 

157 process is under control. 

158 Recommendations: Based on the above, we recommend that for fresh-cut fruits and vegetables: 
 

 

 

 

 

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159 • Microbiological testing of finished product and the environment should play a role in the 

160 verification of control measures. 

161 • Routine testing for E. coli should be conducted and action thresholds established 

162 • Investigative pathogen (e.g., Salmonella, E. coli O157:H7 or other appropriate STEC, L. 

163 monocytogenes) testing when a problem is detected that indicates the potential for the food to 

164 be contaminated with a pathogen 

165 • Depending on commodity, periodic testing of finished product for pathogens (e.g., Salmonella, E. 

166 coli O157:H7 or other appropriate STEC, L. monocytogenes) should be conducted to verify process 

167 control. 

168 • Other activities include annual onsite audits of suppliers and preharvest testing as part of a supply169 chain program; environmental monitoring as a verification of a sanitation program; and robust 

170 process controls and associated verification activities for biological hazards in wash water (i.e., 

171 use of antimicrobials to significantly minimize or prevent the cross-contamination of biological 

172 hazards). 

173 Table E-1. Example of product testing for fresh-cut fruits and vegetables 

Target 

Microorganism 

Microbial 

Limit 

Recommended Action if 

Limit is Exceeded 

Comments 

E. coli <100 CFU/g Investigate reason for 

exceeding limit and correct. 

Determine if pathogen testing 

is warranted. 

Routine testing 
 

 

 

 

 

 

 

 

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Target 

Microorganism 

Microbial 

Limit 

Recommended Action if 

Limit is Exceeded 

Comments 

Salmonella Negative in 

375 g 

Destroy lot. Investigate cause 

of contamination. Determine if 

other lots involved. Determine 

steps to prevent reoccurrence. 

Depending on commodity, 

geographical location and use of 

GAPs; sample size may vary, 

Can composite 15 25g samples 

into one 375 g analytical unit; 

sample numbers should 

increase for investigation 

sampling (e.g., 60 25 g samples 

tested individually or 

composited into 4 375 g 

analytical units) 

E. coli O157:H7 

(or other 

appropriate 

STEC) 

Negative in 

25g 

Destroy lot. Investigate cause 

of contamination. Determine if 

other lots involved. Determine 

steps to prevent reoccurrence. 

Depending on commodity, 

geographical location and use of 

GAPs; sample size may vary. 

Listeria Negative in Destroy lot. Investigate cause Depending on commodity, 

monocytogenes 25g of contamination. Determine if 

other lots involved. Determine 

steps to prevent reoccurrence. 

geographical location and use of 

GAPs; sample size may vary. 

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3. RTE dried/dehydrated fruits 

Examples of dried or dehydrated fruits include cranberries, raisins, apricots, sliced apples, etc. 

The process for creating dehydrated fruits typically involves rinsing and trimming or cutting to the 

appropriate size. Industrial dryers can be used to dehydrate the fruits, or the fruits can be placed on trays 

and dried in the open air and sunlight. Most dried fruits are acidic with a low pH (< 4) and low water 

content. 

Question 1. What principles and criteria should a company apply in determining the need for and in 

designing an effective microbial testing program to verify that processes are effectively controlling 

microbial pathogens? 

The drying process is used to minimize the potential for the presence and growth of pathogens as it 

lowers the pH of the fruit and reduces the water content. The following are considerations to be applied 

in the design of a microbial testing program that includes finished product testing. 

a. The pH of the fruit before drying (acidic fruit), the aw and pH of fully dried fruit, as well as other 

properties that may be unique to a specific dried fruit, such as the antimicrobial properties of 

cranberries. 

b. The quality of the fruit being used may contribute to the anticipated hazard profile; for some fruits, 

bruising or surface injuries may contribute to the microbiological risk of further processing. The 

process of preparing the fruit for drying, for example, whether it is washed, cored, sliced or cubed. 

These processes may also lead to potential for cross-contamination. 

c. Supply chain considerations including growing location and practices, shipment and storage, as well 

as other information gathered under supplier verification activities, such as supplier history, supplier 

controls and testing data. 
 

 

 

 

 

 

 

 

 

 

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197 d. Finally, the end use of the dried fruit may contribute to the microbiological testing program, for 

198 example, dried fruit can be rehydrated and included in baked goods or other products, made into 

199 pastes or consumed as is- all of which may contribute to the types of data that would be expected. 

200 Criteria a facility can apply to determine whether and how often to test ready-to-eat finished 

201 products: 

Criterion/Factor Response RTE dried/dehydrated fruits 

A. Are pathogens associated 

with the food or 

ingredients? 

Yes, but there is variability in the pathogens (hazard) and prevalence 

(likelihood of occurrence) that is dependent on a number of factors 

including commodity, farming system, region and other variable 

events (such as season or weather), poor worker hygiene, harvest or 

post-harvest practices. 

Pathogen (bacterial and parasitic) risk varies also depending on the 

processing and/or drying environment and effectiveness of associated 

process controls. Outside drying activities may have different risks 

than a facility-based dehydration process. 

Post-process contamination resulting from handling in bulk and prior 

to consumer packaging can also be a potential source for pathogens, 

specifically viruses, as dried fruits are often sold in bulk for dispensing 

at the store level and/or in mixtures sold in bulk. 

B. Are the ingredients likely to 

be contaminated? 

Depending on the fruit, its quality, growing practices, transportation 

and storage, the incoming commodities have potential for pathogen 

contamination. Fruits of lower pH are less likely to be contaminated. 
 

 

 

 

 

 

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Criterion/Factor Response RTE dried/dehydrated fruits 

C. Are there robust processing 

control procedures such as 

a kill step or other reduction 

methods/controls? 

Information on the effect of drying on microbial inactivation is limited; 

drying/dehydration can result in microbial inactivation, but this is 

dependent on time, temperature, pathogen, drying technology, and 

type of fruit/vegetable, and more information is needed to validate 

these processes (Bourdoux, Li, Rajkovic, Devlieghere, & Uyttendaele, 

2016). Drying/dehydration is adequate to control microbial growth, 

but pathogens may survive. 

D. Is there a potential for 

recontamination from the 

handling or the 

environment? 

Yes, the product is exposed to the environment during ingredient 

preparation; however, sanitation controls and a robust environmental 

monitoring program (EMP) can reduce the potential to be 

contaminated with microbial pathogens. 

E. Does the product support 

survival or growth? 

Product is not likely to support growth due to low water activity. The 

duration of survival depends on other stressors, such as acidity of the 

fruit and storage temperature; Salmonella can survive for 6-8 months 

at 4°C (1). 

F. Is this product meant for 

higher risk population? 

In most instances the product is being made for the general population 

G.What is the shelf life of the 

product? 

Typically, several years; 1-2 years for select products 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Response RTE dried/dehydrated fruits 

H.Will consumer handling and 

use increase or decrease 

risk of pathogen survival, 

growth, or toxin 

production? 

Bulk containers for storage and dispensing in marketplaces can add 

risk for cross-contamination with viruses. If appropriately dried, the 

fruits are considered shelf-stable and robust for prolonged storage. 

202 

203 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

204 enzymes, would be an appropriate verification activity? 

205 Analytical confirmation of preservatives (such as sulfites) water activity and pH may all be 

206 appropriate tests to be completed during processing and on finished product as a verification activity. 

207 The drying conditions and time should be supported by appropriate measures of control (such as water 

208 activity) that can be applied at several points to ensure final food safety parameters are achieved. 

209 Question 3. Are there situations where (microbial) verification testing would not be necessary if there 

210 is evidence that the appropriate treatment was, in fact, applied? 

211 Yes, if supply chain is well-understood, pH and aw of the finished product do not support pathogen 

212 growth and the monitoring activity of drying or de-hydrate step demonstrated control. However, 

213 sanitation and environmental controls must demonstrate hygienic control of the environment to ensure 

214 there is there no cross contamination with low infectious dose pathogens. 

215 Question 4. When microbial testing is an appropriate verification activity (for finished product), what 

216 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

217 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? What are 

218 appropriate indicator microorganisms for verifying processes adequately control pathogens? 
 

 

 

 

 

 

 

 

 

 

 

 

 

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219 The appropriate microbial testing would vary based on the fruit, the processes for growing, 

harvesting and drying, as well as the final pH and aw. Quantitative indicators of general hygiene may be 

221 considered appropriate at initiation, in-process or end, including APC, Enterobacteriaceae, and/or 

222 coliforms. Molds may also be appropriate for monitoring the general hygiene of the process as well as 

223 the finished good. Some shelf-life surveillance may also be appropriate, again for general indicators of 

224 hygiene. Such testing may also be appropriate if there was an important change in the process, such as 

incoming material changes or new equipment, or to support changes in sanitary controls. 

226 Question 5. What principles and criteria should a company apply in determining the frequency of testing 

227 finished product to determine if the company’s food safety system for that product is effective? 

228 Finished product testing (lot control and/or process control) may be appropriate if there is a change in 

229 supplier, concern regarding and emerging issue, environmental monitoring data demonstrates a trend 

or other seasonality considerations for the fruit/vegetable source which changes the risk profile of the 

231 starting material. 

232 Principles or criteria may include 1) commodity and/or growing/harvesting of a commodity; 2) if 

233 the dehydration or drying process is well-controlled and validated (for example, outdoor dehydrating 

234 process is less controlled than an indoor equipment-based dehydration process); 3) efficacy of sanitation 

programs as evaluated through environmental monitoring 4) or events, such an emerging supply chain 

236 concern, new equipment installation or changes in supplier. 

237 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

238 the goal of verifying the adequacy of control of microbial hazards? 

239 Pathogen testing (pre-harvest or testing at receiving) may be necessary depending on the commodity, if 

there is an emerging issue, a risk associated with the farming or harvesting system (i.e., absence of water 

241 treatment for overhead irrigation) or for a new supplier or change at supplier. Lot acceptance testing 

242 could be considered as the shelf-life allows for this type of testing to be applied. 
 

 

 

 

 

 

 

 

 

 

 

 

 

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Additional points of verification may not eliminate the need for finished product testing but are 

important, including pathogen environmental monitoring and sanitation/hygiene verification testing. 

Question 7: What impact does environmental monitoring have on frequency and extent of product 

testing verification activities by companies? 

Environmental monitoring for pathogens of concern (likely Salmonella and Listeria) is warranted 

if the drying process is conducted in a closed environment and aided by equipment that can facilitate 

cross-contamination. 

However, if the process is an outdoor process such as "sun-drying" then all reasonable 

precautions need to be followed to prevent contamination. Lot acceptance testing may be appropriate 

because of the limitations in deploying an environmental monitoring program and sanitation controls. 

Question 8: What criteria should a company apply in determining that microbial testing results indicate 

a loss of (systemic) process control? What actions should a company take if test results indicate a loss 

of control? When verification testing indicates loss of process control, to what extent should 

verification testing be increased, how far upstream and downstream should it go, and when and how 

should it be scaled back? 

For end products, microbiological testing is not considered a primary means of routinely assessing 

product safety and stability. Assessment of safety is best carried out through assurance that preventive 

controls have been established and executed, as necessary. Microbiological testing can provide a 

supporting role here, to verify hygiene and the drying process and can be reduced based on results 

demonstrating the process is well under control. If significant changes are introduced or if there is a 

failure in the process control, then testing can be intensified temporarily, including finished product 

testing, to verify that the process returns to control. 
 

 

 

 

 

 

 

 

 

 

 

 

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Environmental monitoring and sanitation controls provide key verification/monitoring data to 

266 support that cross-contamination during the drying and storage process are significantly minimized or 

267 prevented. 

268 

269 See Appendix D (Nuts and Nut/Seed Products) Tables D-2 and D-4 for microbiological limits for hygiene 

verification as these limits are appropriate as well for a dry processing facility supporting fruit dehydration. 

271 

272 REFERENCES 

273 1. Beuchat, L. R., and D. A. Mann. 2014. Survival of Salmonella on dried fruits and in aqueous dried 

274 fruit homogenates as affected by temperature. J. Food Prot. 77:1102-1109. 

2. Centers for Disease Control and Prevention. 2021. List of Selected Multistate Foodborne 

276 Outbreak Investigations. https://www.cdc.gov/foodsafety/outbreaks/multistate-outbreaks/outbreaks277 list.html, Accessed 21 Februrary 2021. 

278 3. Gombas, D., Y. Luo, J. Brennan, G. Shergill, R. Petran, R. Walsh, H. Hau, K. Khurana, B. Zomorodi, 

279 J. Rosen, R. Varley, and K. Deng. 2017. Guidelines to validate control of cross-contamination during 

washing of fresh-cut leafy vegetables. J. Food Prot. 80:312-330. 

281 4. International Commission on Microbiological Specifications for Foods. 1996. Microorganisms in 

282 foods 5: characteristics of microbial pathogens. Blackie Academic & Professional, New York. 

283 5. International Commission on Microbiological Specifications for Foods. 2005. Microorganisms in 

284 Foods 6: Microbial ecology of food commodities, 2nd ed. Kluwer Academic/Plenum Publishers, New 

York. 

286 6. International Commission on Microbiological Specifications for Foods., and F. S. Thatcher. 1978. 

287 Microorganisms in foods 1 : their significance and methods of enumeration : a publication of the 
 

 

 

 

 

 

 

 

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295

300

305

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288 International Commission on Microbiological Specifications for Foods (ICMSF) of the International 

289 Association of Microbiological Societies. University of Toronto Press, Toronto ; Buffalo. 

7. Marshall, K. E., A. Hexemer, S. L. Seelman, M. K. Fatica, T. Blessington, M. Hajmeer, H. 

291 Kisselburgh, R. Atkinson, K. Hill, and D. Sharma. 2020. Investigations of Shiga Toxin-Producing 

292 Escherichia coli Outbreaks Linked to Leafy Greens, United States and Canada, 2009–2018. Emerg Infect 

293 Dis. 26:2319-2328. 

294 8. National Advisory Committee for Microbiological Criteria for Foods. 2018. Response to 

questions posed by the Department of Defense regarding microbiological criteria as indicators of 

296 process control or insanitary conditions. J. Food Prot. 81:115-141. 

297 9. U.S. Food and Drug Administration. 2015. FSMA Final Rule on Produce Safety: Standards for the 

298 growing, harvesting, packing, and holding of produce for human consumption. 

299 https://www.fda.gov/food/food-safety-modernization-act-fsma/fsma-final-rule-produce-safety 

Accessed 14 April 2021. 

301 10. U.S. Food and Drug Administration. 2021. Outbreaks of Foodborne Illness. 

302 https://www.fda.gov/food/recalls-outbreaks-emergencies/outbreaks-foodborne-illness Accessed 14 

303 April 2021. 

304 11. U.S. Food and Drug Adminstration. 2017. Control of Listeria monocytogenes in Ready-To-Eat 

Foods: Guidance for Industry. https://www.fda.gov/files/food/published/Draft-Guidance-for-Industry--

306 Control-of-Listeria-monocytogenes-in-Ready-To-Eat-Foods-%28PDF%29.pdf, Accessed April 26, 2021. 

307 12. Western Growers Association. 2020. CA LGMA 2020 Commodity specific food safety guidelines 

308 for lettuce and leafy greens supply chain. https://www.leafygreenguidance.com/wp309 content/uploads/2020/10/CA-LGMA-Metrics-August-2020_Final_Clean_9-15-20.pdf, Accessed April 14, 

2021. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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1 APPENDIX F - CATEGORY: SPICES AND HERBS 

2 Introduction 

3 Spices and herbs are dried fragrant, aromatic or pungent edible plant substances in whole, 

4 broken, or ground form. They are differentiated based on the part of the plant from which they originate. 

5 Spices originate from seeds (e.g., cumin, sesame, poppy), leaves (e.g., basil, mint), roots (e.g., turmeric, 

6 ginger), bark (e.g., cinnamon), or flower/flower parts (e.g., saffron, cloves). Herbs, or culinary herbs, are 

7 defined as originating from non-woody plants, e.g., tarragon (22, 23). 

8 Spices, spice blends, and herbs have been implicated in foodborne illness outbreaks, despite 

9 having a low water activity (less than 0.85). The following biological hazards for both untreated/raw and 

10 treated herbs and spices are listed in FDA’s draft Appendix 1: Bacillus cereus, Clostridium botulinum, C. 

11 perfringens, pathogenic E. coli, and Salmonella spp. (21). Salmonella spp. and B. cereus are implicated in 

12 the majority of the outbreaks (24, 25). Agricultural conditions play a significant role in product microbial 

13 contamination (1). The process of drying spices and herbs allows some pathogens, such as Salmonella spp. 

14 and B. cereus, to survive for an extended period of time (10, 19). When contaminated spices or herbs in 

15 untreated or treated form are added to ready-to-eat foods without further processing, the food has the 

16 potential to become a vehicle for foodborne illness. 

17 Source plants for spices and herbs are grown worldwide. Agricultural practices vary widely by 

18 country and within a country. Farms may be basic or highly mechanized. The drying process may be rapid 

19 by mechanical means or by natural sunlight over several days (8). The supply and processing chain can 

20 range from very simple within one processing facility to complex with numerous stages including 

21 outsourcing to a third party for pathogen reduction treatments (e.g., irradiation). The potential for 

22 contamination can occur at any stage: growing, harvesting, processing, packaging, storage, and 

23 distribution (8). 

24 
 

 

 

 

 

 

 

 

 

 

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25 Spices and spice blends may or may not be further processed for lethality. Microbial reduction 

26 treatments include steam treatments, ethylene oxide or other gas treatments, or irradiation. 

27 

28 Figure 1. Basic flow chart of spice processing. 

Microbial 

Reduction 

Treatment 

Raw Spice Drying Sorting and 

Cleaning 

Blending 

Bulk 

Package 

Final 

Package 

Grinding 

29 

30 

31 Optional steps in the process are shown with broken lines. Some processes may include a 

32 microbial (pathogen) reduction treatment, some may include blending, and some may include both 

33 intervention and blending steps. 

34 The intrinsic properties of certain spices and herbs such as allspice, cinnamon, cloves, and oregano 

35 can interfere with laboratory testing methodologies (6). Microbial inhibition and the impact on detection 

36 is addressed in the FDA’s Bacteriological Analytical Manual (BAM) (4). The recommended approach to 

37 alleviating inhibition by spices containing inhibitory compounds is to dilute them with an initial 1:100 or 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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38 1:1000 dilution rather than the standard 1:10 dilution. However, as noted in the BAM, it is not possible 

39 to completely neutralize the toxicity of some spices, and this does affect the ability to recover pathogens 

40 contaminants, especially at low levels. 

41 

42 Spice Products 

43 RTE spices and spice blends, not processed for lethality 

44 RTE spices and spice blends, processed for lethality 

45 Dried, chopped herbs 

46 1. RTE Spices and Spice Blends, Not Processed for Lethality1 

47 Raw RTE spices are more likely to be contaminated with Salmonella (26) than spices processed 

48 with microbial reduction treatments. Some spices/herbs are not processed with a pathogen reduction 

49 treatment because the available treatments can have an adverse impact on quality. Dehydrated onion 

50 and garlic are examples. Pathogen hazards can originate with the growers. Soil, organic fertilizers, 

51 compost and water are sources of microorganisms that remain with the spice after drying. 

52 Manufacturers of RTE raw spices and spice blends must implement preventive controls to 

53 significantly minimize or prevent hazards and ensure that the RTE raw spices they manufacture will not 

54 be adulterated. These preventive controls include Good Agricultural Practices (GAPs) and sanitation 

55 controls on the part of their suppliers (i.e., farms growing, harvesting, packing, and holding the raw 

56 spices), supplier audits as part of their supply-chain program, and sanitation controls within their own 

57 facilities. While these interventions can reduce risk, they may not be sufficient, as process controls may 

58 not be adequate to completely eliminate the hazard. In 2019, the U.S was the top importer of spices 

1 Many spices that have pathogen hazards that are not processed to control such hazards may be destined for use 

in processed products that are subjected to processes such as cooking that will control the hazard; these are 

considered non-ready-to-eat (NRTE) spices and are not covered in this Appendix F. NRTE spices are required to 

bear a disclosure that they are not processed to control microbial pathogens. 
 

 

 

 

 

 

 

 

 

 

 

 

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59 globally (7, 17). A robust supply chain control program is essential for imported spices where visibility to 

good harvesting and sanitary practices during handling and storage may be limited. 

61 An example of an unprocessed imported spice sold as RTE is saffron derived from crocus 

62 flowers. Saffron is not thermally processed or irradiated in order to preserve the color, taste, and odor 

63 of the product (9). Most saffron is used in cooked dishes and no recalls or outbreaks associated with 

64 saffron have been recorded to date. However, one study found high microbial loads in saffron grown in 

Iran. The study could not rule out the possibility that poor harvesting and lack of sanitary practices 

66 during storage contributed to elevated microbial levels (18). How and where spices are stored have 

67 been related to the level of contamination. Spices held in bulk have higher concentrations of pathogens. 

68 Unpacked spices, stored in bulk open containers, can be contaminated through dust, wastewater and 

69 animal/human excreta (18). 

71 Question 1. What principles and criteria should a company apply in determining the need for and in 

72 designing an effective microbial testing program to verify that processes are effectively controlling 

73 microbial pathogens? 

74 A hazard analysis is required considering (1) possible microbial hazards, (2) likelihood of 

occurrence, (3) available processing control procedures such as a kill step or other reduction 

76 methods/controls, (4) potential for inherent contamination or recontamination after processing from 

77 handling or the environment, (5) survival (persistence) or growth on the product, (6) intended consumer, 

78 (7) shelf life of the product, and (8) steps in the process where testing would be appropriate to verify food 

79 safety controls. 

A testing program applied to a spice or spice blend not processed for lethality should focus on 

81 the potential presence of a pathogen. Note that testing alone will not provide adequate assurance of 
 

 

 

 

 

 

 

 

 

 

 

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82 safety for most untreated spices; when pathogens such as Salmonella have been associated with a RTE 

83 spice, it should be treated to reduce the risk. 

84 

Criterion/Factor Response 

1. Are pathogens associated 

with the food or 

ingredients? 

Yes – pathogenic bacteria are associated with raw unprocessed spices 

(21, 24-27) 

2. Are the ingredients likely to 

be contaminated? 

Yes. A supplier verification program is necessary but may not be 

sufficient to control the hazard. Spices sold at retail in the U.S are 

more likely to be treated to mitigate a hazard (26). 

3. Are there robust processing 

control procedures such as 

a kill step or other reduction 

methods/controls? 

No. 

4. Is there a potential for 

recontamination from the 

handling or the 

environment? 

Yes. Manufacturing environments in facilities that handle raw spices 

and blends may be a source of contamination. 

5. Does the product support 

survival or growth? 

Pathogens will survive but not grow. 

6. Is this product meant for 

higher risk population? 

The product is made for the general population including high risk 

consumers. 
 

 

 

 

 

 

 

 

 

 

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Criterion/Factor Response 

7. What is the shelf life of the 

product? 

1 – 2 years 

Will consumer handling and 

use increase or decrease 

risk of pathogen survival, 

growth, or toxin 

production? 

The risk for outgrowth may be increased if the spice or blend is added 

to a product with a water activity that allows outgrowth. 

Pathogen risk is reduced if the spices or blends are added to a recipe 

that is cooked. 

85 

86 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

87 enzymes, would be an appropriate verification activity? 

88 There are no situations where testing other than for pathogens or indicator organisms would be 

89 appropriate. 

90 

91 Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

92 evidence that the appropriate treatment was, in fact, applied? 

93 No. Raw RTE spices and spice blends are not processed for lethality by definition. Good 

94 Agricultural Practices are relied upon for limited control. Finished product testing for Salmonella in the 

95 absence of a pathogen mitigation process is used to screen for pathogen contamination. 

96 

97 Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

98 considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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99 specific indicator organism) and type of test (e.g., presence/absence or enumeration)? What are 

appropriate indicator microorganisms for verifying processes adequately control pathogens? 

101 The primary pathogen of concern in raw spices and spice blends is non-typhoidal Salmonella. The 

102 presence of this pathogen in ready-to-eat spices and spice blends is considered adulteration. 

103 Manufacturers should test in-process and finished products routinely for Aerobic Plate Counts 

104 (APC) organisms, coliforms and Salmonella; lot by lot is recommended. Manufacturers should routinely 

test the environment for Salmonella. Nonroutine testing of finished products by manufacturers, when 

106 deemed necessary, includes E. coli and E. coli O157:H7 (or other STEC as appropriate) (NACMCF 2018). 

107 

108 Question 5. What principles and criteria should a company apply in determining the frequency of testing 

109 finished product to determine if the company’s food safety system for that product is effective? 

A sampling plan and evaluation criteria should be developed based on the type of spice or spice 

111 blend, the history of foodborne illness outbreaks associated with the spice or spice blend, the source of 

112 the raw spices used in manufacture, the grower’s history and GAP programs, the storage and 

113 distribution chain and the risk for cross contamination from the manufacturer’s process environment. 

114 The sampling plan should clearly define what is considered the “lot”. 

Finished product testing for Salmonella using FDA category II sampling is recommended on a per 

116 lot basis to screen for pathogen contamination in the absence of a pathogen mitigation process. FDA 

117 Category II includes foods that would not normally be subjected to a process lethal to Salmonella between 

118 the time of sampling and consumption (FDA, 2018). The parameters of Category II are: 30 analytical units/ 

119 25-gram samples. The samples may be aggregated into 375-gram analytical units. 

A robust EMP that includes testing for Salmonella is essential. A weekly program is 

121 recommended. 

122 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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123 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

124 the goal of verifying the adequacy of control of microbial hazards? 

Manufacturers of untreated RTE spices and spice blends do not apply a microbial reduction 

126 treatment to the bulk shipments of raw spices they receive and process. Testing at the end of the 

127 process is performed as a screen for the presence of Salmonella and does not verify the adequacy of 

128 microbial hazard controls that are not in place. An inbound testing program for the presence of 

129 Salmonella and populations of microbes on APC agar using a statistically significant sampling plan 

provides another level of screening for ingredient contamination prior to production. 

131 

132 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

133 product testing verification activities by companies? 

134 Although the primary concern might be the presence of pathogens in the actual spice, microbial 

contamination from the environment must also be considered. In a facility manufacturing product using 

136 raw spices, environmental pathogens such as Salmonella will be introduced to the plant environment 

137 from these raw ingredients. Sanitation controls will be needed to prevent Salmonella from gaining a 

138 harborage in the facility whereby it could contaminate products. A robust EMP that includes testing for 

139 Salmonella is essential. 

141 Question 8: What criteria should a company apply in determining that microbial testing results indicate 

142 a loss of (systemic) process control? 

143 What actions should a company take if test results indicate a loss of control? 

144 When verification testing indicates loss of process control, to what extent should verification testing be 

increased, how far upstream and downstream should it go, and when and how should it be scaled back? 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Testing directly for the presence of Salmonella in inbound bulk ingredients and finished products 

does not verify an effective pathogen mitigation process in the case of untreated RTE spices and blends. 

The detection of Salmonella should cause inbound ingredients to be rejected and finished products to 

be destroyed or reprocessed using a validated pathogen mitigation treatment. 

In the absence of positive pathogen results, out of specification indicator organism test results 

could also signal inherent contamination from the field or that the materials had been subjected to 

unhygienic conditions in the manufacturing facility (Table F-1). However, high populations enumerated 

on APC agar do not necessarily correlate with pathogen risk. Organic products, for example, may have 

higher plate counts due to agricultural methods used. Higher APC counts could trigger more robust 

pathogen testing and a review of controls. If warranted, a lethality treatment, if applicable, could be 

applied. 

Of note, spices can contain high levels of sporeformers, which can result in high APC 

populations. These may include pathogenic sporeformers, which at levels <104 have not been 

associated with a safety risk when the spices are dry. However, the presence of microbial spores may be 

a concern for the foods in which the spices are used, and customers may request that treatments to 

reduce bacterial spores and/or that spices be tested for certain types of spores based on the intended 

use of the spice. 
 

 

 

 

 

 

 

 

 

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165 Table F-1. Microbial targets, limits, and recommended actions if limits are exceeded for RTE spices and 

166 spice blends, not processed for lethality. 

Target 

Microorganism in 

Finished Product 

Microbiological Limit Recommended Action if 

Limit is Exceeded 

Comments 

Aerobic Plate Count 

(APC) 

<106 CFU/g 

Depending on the specific 

spice and geographic source, 

exceeding this limit may 

require appropriate 

investigative and corrective 

actions. 

Coliforms <104 CFU/g 

Investigate, implement 

corrective action 

Option: Test for 

generic E. coli (<10 

CFU/g) (Nonroutine) 

Salmonella Negative in 2 X 375-g 

samples 

Divert for reprocessing, if 

appropriate, or reject. 

Investigate and implement 

corrective action 

Two 375-g analytical 

units derived from 30 

x 25-g samples 

E. coli (O157:H7 or 

other STEC) 

Negative in 25 g 

Divert for reprocessing, if 

appropriate, or reject. 

Investigate and implement 

corrective action 

Investigative testing 

if coliform or generic 

E. coli or 

environmental 
 

 

 

 

 

 

 

 

 

 

 

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Target 

Microorganism in 

Finished Product 

Microbiological Limit Recommended Action if 

Limit is Exceeded 

Comments 

testing suggest 

contamation 

167 Adapted from: Appendix J. Table J.40 (16). 

168 

169 2. RTE spices and spice blends, processed for lethality 

170 These spices and spice blends have undergone a microbial reduction treatment. Some lethality 

171 (intervention) processes are performed by third party contractors, while others are performed by the 

172 company. 

173 

174 Question 1. What principles and criteria should a company apply in determining the need for and in 

175 designing an effective microbial testing program to verify that processes are effectively controlling 

176 microbial pathogens? 

177 A hazard analysis is required as detailed in the “Raw RTE Spices and Spice Blends Not Processed 

178 for Lethality” section of this Appendix F. However, the complexity of the manufacturing process for 

179 processed spices and blends must be considered when determining risk, assigning pathogen controls 

180 across the manufacturing continuum and designing a testing program to verify the effectiveness of 

181 controls implemented. There may be two or more entities taking part in the overall manufacturing 

182 process. 

183 As an example, one entity could be a business that makes treated spices (they may treat them 

184 in-house or they may use a contract sterilizer). This entity could be a supplier to a business that makes 
 

 

 

 

 

 

 

 

 

 

 

 

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185 spice blends. A spice blend manufacturer could receive treated spices for blending or they could receive 

186 untreated spices and treat them before or after blending. While blending treated spices they receive 

187 from a supplier may be most common, this type of context is important in determining the testing that 

188 could apply at each stage of the process. As an example, a processor of an individual spice may test the 

189 spice after a microbial reduction treatment has been applied. The individual spice may be purchased by 

190 a spice blend manufacturer, who may test the individual spices upon receipt as a verification activity, 

191 and then test the finished blend of spices prior to shipment. 

192 If a spice or spice blend is processed using a validated microbial reduction treatment, the 

193 primary focus of microbial testing at the finished product manufacturer should be verification that the 

194 process preventive control was successfully applied and that practices within their facility prevent cross 

195 contamination from the processing environment. The same verification program could be used at the 

196 contract sterilizer and verified by the manufacturer as part of their supplier assurance program. 

Criterion/Factor Response 

1. Are pathogens associated 

with the food or ingredients? 

Yes – pathogenic bacteria are associated with raw unprocessed spices 

(24, 25) 

2. Are the ingredients likely to 

be contaminated? 

Yes, and supplier verification program is necessary for some 

ingredients. Each ingredient needs to be assessed. 

3. Are there robust processing 

control procedures such as a 

kill step or other reduction 

methods/controls? 

Yes – spices and herbs may be subjected to lethal treatments such as 

with gas, steam, ionizing radiation, or other processes. 
 

 

 

 

 

 

 

 

 

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Criterion/Factor Response 

4. Is there a potential for 

recontamination from the 

handling or the environment? 

Yes - unless the product is treated in package. Where spices and 

blends are treated in boxes or bags by ionizing radiation, ETO or steam, 

there is limited exposure (if any). If treated products are subsequently 

repackaged (e.g., in jars) then the potential for exposure exists. 

5. Does the product support 

survival or growth? 

Pathogens will survive but not grow. 

6. Is this product meant for 

higher risk population? 

In most instances the product is being made for the general 

population. 

7. What is the shelf life of the 

product? 

1 – 2 years 

8. Will consumer handling and 

use increase or decrease risk 

of pathogen survival, growth, 

or toxin production? 

Consumer handling may not have an impact on risk. In some cases, 

consumer handling may reduce risk if the spices are added to a recipe 

which is heated. However, the risk may be increased if spices 

containing pathogenic sporeformers are used in a food that is held 

under conditions that allow growth. 

197 

198 Question 2. Are there situations in which testing other than for pathogens or indicator organisms, e.g., 

199 enzymes, would be an appropriate verification activity? 

200 No. However, if the process intervention/ microbial reduction treatment is validated, and the 

201 process is monitored and verified, routine microbial testing of the finished product may not be 

202 necessary if the product is treated in an enclosed container and not exposed to the environment. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

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Question 3. Are there situations where [microbial] verification testing would not be necessary if there is 

evidence that the appropriate treatment was, in fact, applied? 

As noted above, if the process intervention/ microbial reduction treatment is validated and 

applied to a packaged product, routine microbial testing of the finished product for pathogens may not 

be necessary or may be limited. The process should conform to the appropriate ISO or ASTM standards 

(e.g., ISO 14470 (13) or ASTM F1885-4 (5) for irradiation or ISO 11135 (14) for ethylene oxide). Steam 

processes are typically proprietary. 

However, if there is still a concern for post-process contamination from the environment, as 

would occur when the product is processed then subsequently packaged, microbial verification testing 

for Salmonella and Enterobacteriaceae is recommended. 

Question 4. When microbial testing is an appropriate verification activity [for finished product], what 

considerations should a company apply in selecting the test microorganism (e.g., specific pathogen or 

specific indicator organism) and type of test (e.g., presence/absence or enumeration)? 

What are appropriate indicator microorganisms for verifying processes adequately control pathogens? 

Since these spices and herbs have been given a lethality treatment, the test organisms should be 

those that verify process delivery. Testing for Enterobacteriaceae or coliforms and Salmonella is useful 

to verify process control (11). The surviving population of Enterobacteriaceae or coliforms should be 

determined quantitatively, using a method which has been demonstrated to recover injured 

microorganisms (15). Salmonella should be absent. 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

225

230

235

240

245

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Question 5. What principles and criteria should a company apply in determining the frequency of testing 

226 finished product to determine if the company’s food safety system for that product is effective? 

227 Finished product testing may not be warranted (or may be limited) for validated processes verified 

228 to be under control; however, it is incumbent upon the manufacturer of treated spices and blends to 

229 determine if finished product testing for pathogens or indicator organisms would provide information 

useful to assess process control in their facility. Factors to consider when deciding to conduct finished 

231 product testing include who is doing the treatment, how rigorous is the treatment, process validation 

232 information, confidence in the entity doing the treatment, historical information from the supplier, how 

233 much exposure to the environment and other factors. It is not uncommon for treated spice 

234 manufacturers to conduct finished product testing and supply a certificate of analysis with the products 

they ship. 

236 When finished product is screened for pathogens and/or coliforms or Enterobacteriaceae, 

237 testing a representative sample from a complete process run provides the most information about 

238 process control. A sampling plan and evaluation criteria should be risk based on the type of spice, the 

239 type of process, the geographic source of the spice and prior history. Sampling plans should be risk 

based and incorporate the fundamental principles of statistical process control and trend analysis. 

241 Upper control limits should be established for quantitative analyses, with action levels determined as 

242 some point less than the upper control limit. Sampling plans should follow the basic guidelines of 

243 investigative, routine and reduced sampling. 

244 FDA guidance provides an example of a sampling plan (sample size and number) when testing 

for Salmonella in foods that would not normally be subjected to a process lethal to Salmonella between 

246 the time of sampling and consumption (2, 3, 20). The sampling parameters are 30 analytical units/ 25-

247 gram samples. The samples may be aggregated into 375-gram analytical units. ICMSF provides 

248 additional information about finished product sampling plans commensurate with risk (12). 
 

 

 

 

 

 

 

 

 

 

 

 

250

255

260

265

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249 Question 6: Are there situations in which testing at sites other than the end of the process can achieve 

the goal of verifying the adequacy of control of microbial hazards? 

251 For treated RTE spices and herbs there would be no purpose in testing prior to a microbial 

252 reduction step. Testing after the treatment and prior to packaging could be appropriate when 

253 combined with effective GMPs and sanitation controls to prevent recontamination and with 

254 environmental monitoring. 

256 Question 7: What impact should (does) environmental monitoring have on frequency and extent of 

257 product testing verification activities by companies? 

258 During and after treatment, spices and herbs are usually in a container that limits environmental 

259 exposure. If the spices and herbs are repackaged after treatment, then an environmental monitoring 

program may be appropriate. An environmental monitoring program may result in a temporary 

261 movement to investigational sampling, including testing for Salmonella, when an event in the 

262 environmental monitoring program indicates a potential for contamination of the spice or herb. 

263 

264 Question 8: What criteria should a company apply in determining that microbial testing results indicate 

a loss of (systemic) process control? 

266 A loss of systemic process control is indicated when a pathogen is detected in finished product or 

267 indicator data repeatedly exceed the limits established for a stable process operating within predictable 

268 process variation or exceptionally high indicator levels are observed. 

269 Producers of RTE spices/blends processed for lethality rely on preventive controls that include 

validated pathogen mitigation treatments, sanitation, and supply-chain programs. The finding of a 

271 positive pathogen result could indicate a loss in sanitation control. When a pathogen is detected from a 

272 sample taken at the end of the production line, the recommended action is to reject a lot of spices 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

275

280

285

290

295

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273 represented by the sample unless reprocessing using a validated treatment can be conducted. The 

274 repeated finding of indicator organisms such as coliforms or Enterobacteriaceae above a threshold level 

can also indicate a loss of sanitation control although actions taken would follow a tiered approach based 

276 on numbers and frequency of occurrence. 

277 Table F-2 details recommended specification limits for Salmonella, coliforms and 

278 Enterobacteriaceae that verify controls are in place and effective within the manufacturing facility. Note 

279 that treated spices can contain high levels of sporeformers which can result in high APC counts. As an 

example, black peppercorns treated with ethylene oxide can still have an APC of approximately 106 

281 CFU/gram with no detectable surviving Gram-negative bacteria. 

282 

283 What actions should a company take if test results indicate a loss of control? 

284 Depending on the seriousness of the hazard, the types of actions that companies may take when loss 

of control is indicated by microbial testing results include: 

286 1. Verification of the sanitation program 

287 2. Verification that processing parameters were met 

288 3. Increase verification testing frequency 

289 4. Stop processing line until a root analysis is completed 

5. Investigate the source of contamination under reduced production 

291 There is no acceptable incidence or population of Salmonella in ready-to-eat spices or herbs. The 

292 detection of Salmonella should cause finished products to be destroyed or reprocessed using a validated 

293 pathogen mitigation treatment. U.S. Regulations prohibit the irradiation of products which have been 

294 previously irradiated. 
 

 

 

 

 

 

 

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296 When verification testing indicates loss of process control, to what extent should verification testing be 

297 increased, how far upstream and downstream should it go, and when and how should it be scaled back? 

298 Root cause analysis will determine whether verification testing should be increased and how 

299 long amplified testing should occur. 

300 

301 Table F-2. Microbial targets, limits, and recommended actions if limits are exceeded for RTE spices and 

302 spice blends, processed for lethality. 

Target 

Microorganism in 

Finished Product 

Microbiological 

Limit 

Recommended Action if 

Limit is Exceeded 

Comments 

Aerobic Plate Count <105 CFU/g Depending on the specific 

spice and geographic source, 

exceeding this limit may 

require appropriate 

investigative and corrective 

actions. 

Spices can contain high 

levels of sporeformers, 

which can result in high 

APC counts even in 

treated spices. 

Coliforms <10 CFU/g Investigate, implement 

corrective action 

Option: Test for generic 

E. coli (<1 MPN/g) 

(Investigative) 

Enterobacteriaceae <102 CFU/g Investigate, implement 

corrective action 
 

 

 

 

 

 

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Salmonella Negative in two 

375 g samples 

Divert for reprocessing, if 

appropriate, or reject. 

Investigate and implement 

corrective action 

Two 375 g analytical 

units derived from 30 

25 g samples 

E. coli (O157:H7 or 

other STEC) 

Negative Divert for reprocessing, if 

appropriate, or reject. 

Investigate and implement 

corrective action 

Investigative testing, if 

EMP or 

coliform/Enterobacteri 

aceae exceed limits 

303 

304 3. Dried, chopped herbs 

305 The responses and criteria for RTE spice and spice blends, not processed for lethality would be 

306 applicable for dried, chopped herbs. Approved suppliers for source plants who follow Good Agricultural 

307 Practices should be used and GMPs adhered to throughout the entire processing, storage, and 

308 distribution chain. The same finished product testing criteria are recommended as shown in Table F1 

309 and F2, depending on whether they have been treated for lethality. 

310 

311 REFERENCES 

312 1. American Spice Trade Association. 2017. Clean, Safe Spices: Guidance from the Amercian Spice 

313 Trade Association. 

314 2. Andrews, W. H., and T. Hammack. 2003. BAM Chapter 1, Food Sampling and Preparation of 

315 Sample Homogenate. In, FDA Bacteriological Analytical Manual, https://www.fda.gov/food/laboratory316 methods-food/bam-chapter-1-food-samplingpreparation-sample-homogenate. 
 

 

 

 

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