FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 1 of 98

For the most current and official copy, check QMiS. 

Sections in This Document 

Purpose .................................................................................................................................... 2

Scope ....................................................................................................................................... 2

Responsibility............................................................................................................................ 3

Background............................................................................................................................... 3

References ............................................................................................................................... 4

Procedure ................................................................................................................................. 4

6.1. Fundamentals ................................................................................................................ 4

6.1.1. Media, Reagent, and Supply Preparation ........................................................ 5

6.1.2. Safety and Hazardous/Infections Waste .......................................................... 6

6.1.3. Quality Assurance ............................................................................................ 8

6.1.4. Polymerase Chain Reaction (PCR) ................................................................. 9

6.1.5. Establishment Inspections ............................................................................. 11

6.2. Basic (Tier 1, i.e. analyses required for analyst level 1 certification) ........................... 12

6.2.1. Salmonella ..................................................................................................... 13

6.2.2. Listeria monocytogenes ................................................................................. 15

6.2.3. Escherichia coli .............................................................................................. 17

6.2.4. Enterohemorrhagic E.coli............................................................................... 19

6.2.5. Staphylococcus aureus .................................................................................. 20

6.2.6. Coliforms ........................................................................................................ 21

6.2.7. Aerobic Plate Count ....................................................................................... 22

6.2.8. Yeast and Mold Count ................................................................................... 23

6.3. Intermediate (Tier 2) ..................................................................................................... 24

6.3.1. Vibrio .............................................................................................................. 25

6.3.2. Bacillus cereus ............................................................................................... 26

6.3.3. Campylobacter ............................................................................................... 27

6.3.4. Yersinia enterocolitica .................................................................................... 28

6.3.5. Clostridium perfringens .................................................................................. 29

6.3.6. Clostridium botulinum .................................................................................... 30

6.3.7. Alkaline Phosphatase .................................................................................... 31

6.3.8. Canned Food and Canned Seam Examination ............................................. 32

6.3.9. Cronobacter spp. (NEW) ................................................................................ 34

6.3.10. Cosmetic Analysis .......................................................................................... 34
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 2 of 98

For the most current and official copy, check QMiS. 

6.3.11. Nutritional and Dietary Supplements (NEW) .................................................. 36

6.4. Advanced (Tier 3) ......................................................................................................... 37

6.4.1. Viruses ........................................................................................................... 37

6.4.2. Cyclospora (NEW) ......................................................................................... 40

6.4.3. Whole Genome Sequencing (WGS) (NEW) .................................................. 41

6.4.4. Select Agents ................................................................................................. 43

Document History ................................................................................................................... 45

Change History ....................................................................................................................... 46

Attachments ............................................................................................................................ 48

Attachment A - Answer Key .................................................................................................... 49

 Purpose 

The purpose of this document is to provide training guidance to new hire 

analysts responsible for performing microbiological analysis of food and feed 

samples and their trainers. This information is both a supplement to local onthe-job training (OJT) and formal classroom training (OTED courses), but also 

provides the standardized framework and defines the minimal requirements for 

local OJT. 

This training chapter introduces the trainee to four categories of 

microbiological analysis: Fundamentals, Basic (Tier 1), Intermediate (Tier 2), 

and Advanced (Tier 3). 

The overall purpose of this chapter is to: 

 Train the analyst to think as a regulatory microbiologist. 

 Introduce typical analytical procedures a regulatory microbiologist should 

know, understand, and may encounter throughout their career. 

 Show where and how the work performed fits into the regulatory framework 

including: reason for sample collection; procedures of an inspection and 

sample collection; sample analysis procedures and approaches; and 

regulatory follow up actions and relationship of items to the FD&C Act. 

 Scope 

This document applies to all FDA regulatory microbiologists involved in the 

analysis of microbiological food and feed samples. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 3 of 98

For the most current and official copy, check QMiS. 

The FDA microbiologist is well termed a regulatory microbiologist because 

everything he or she does is related to the regulation of products and 

manufacturers under the jurisdiction of the Federal Food, Drug, and Cosmetic 

Act (FD&C Act) and Related Acts. Thus, results of his or her work impacts 

directly on those products regulated, and therefore on the consumer. The FDA 

microbiologist can also be called a public health microbiologist as removing 

contaminated food and feed products from the market directly impacts the 

health and welfare of consumers. 

 Responsibility 

3.1. Trainer 

A. Coordinates training with Supervisor(s) and Trainee(s). 

B. Works with new hire Trainee(s) to ensure completion of all basic training 

needed to meet their new hire level 1 analyst certification. 

C. Works with Trainee(s) to ensure completion of all intermediate and 

advanced training needed to meet their regulatory responsibilities. 

3.2. Trainee 

A. Completes required training within specified timeframes. 

B. Reports training received and provides documentation of completed 

training to supervisor. 

C. Reads and complies with standards, regulations, policies, procedures, 

and work instructions. 

3.3. Supervisor and Management 

A. Implements and reviews training records. 

B. Designates local Trainers and ensures that the required and appropriate 

content is covered. 

3.4. Quality Management 

A. Maintains employee competencies matrix records for staff. 

Background 

Food and feed microbiological analyses range from the relatively simple to the 

most complex. In recent years, microbiologists have transitioned from sole use 

of conventional methods to a coupling of state-of-the-art rapid methods with 

the traditional. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 4 of 98

For the most current and official copy, check QMiS. 

During the first year, there will be several basic training courses offered by 

OTED. Training will also be supplemented by computer-based modules 

provided online by ORA U. However, most instruction will come from the 

laboratory. 

Microbiology methodology is an ever-improving science. Training will be a 

continuing process throughout the microbiologist’s career. Future training will 

reinforce and amplify what the trainee has learned. 

References 

B. American Society for Microbiology Laboratory Protocols (background 

discussion on Gram stain, serial dilutions, ELISA, PCR) 

C. Center for Disease Control and Prevention Microbiology Series (free 

training modules on PPE, basic culture media, microscopy, core 

microbiology skills, and molecular techniques) 

D. AOAC Official Methods of Analysis

E. FDA Bacteriological Analytical Manual (BAM) 

F. United States Pharmacopeia (USP) Note: to access USP references 

throughout this document go to Account Information (sharepoint.com) to 

create an FDA library account if you have not already done so. 

 Procedure 

6.1. Fundamentals 

At the foundation of an FDA regulatory microbiologist are the most 

fundamental microbiology skills and techniques. This section will focus on the 

fundamentals of media and reagent preparation, laboratory safety and 

hazardous waste handling, quality assurance and management, and 

polymerase chain reaction. 

The new hire shall also have a basic understanding of the concepts of aseptic 

technique, serial dilutions, quadrant streaking, Gram staining, ELISA tests, and 

other molecular methodology. These topics are covered in more depth as the 

new hire progresses through the Basic category analyses. However, there are 

links for supplemental review in the references section from ASM and CDC. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 5 of 98

For the most current and official copy, check QMiS. 

6.1.1. Media, Reagent, and Supply Preparation 

6.1.1.1. Objective 

A. Familiarize the trainee with preparing, dispensing, and sterilizing 

microbiological media, reagents, and supplies. 

B. Familiarize the trainee with media quality assurance procedures and 

documentation processes. 

C. Familiarize the trainee with safety concerns, such as autoclave safety 

and weighing of powders. 

6.1.1.2. Assignment 

A. The trainer will discuss and demonstrate the equipment used in media 

preparation such as balances, stirrers, dispensers, pH meters, and 

autoclaves. 

B. The trainer will discuss proper personal protective equipment (PPE) to 

be used during each step of media preparation. 

C. The trainee will prepare several kinds of media and buffer solutions, 

representative of the types generally used. These include, but are not 

limited to, selective and non-selective enrichment broths, plating media, 

and phosphate buffer. Examples include Lauryl sulfate tryptose broth, 

Brilliant green lactose broth, EC broth, Lactose broth, Tetrathionate 

broth, Plate count agar, Butterfields Phosphate diluent, Triple sugar iron 

agar, EMB agar, Hektoen enteric agar, Bismuth sulfite agar. 

D. The trainee will prepare and sterilize supplies such as empty bottles and 

tubes, polyvinylchloride tubing, spoons etc. as dictated by individual lab 

use. 

E. The trainee will demonstrate the ability to dispense media, either using 

a manual pipettor, automated pipettor/dispensing system, or both. 

F. The trainer will discuss the function of the components that comprise 

the commonly used media. 

G. The trainer will discuss storage and shelf life of prepared and 

dehydrated media/reagents. 

6.1.1.3. Questions 

A. Which media should not be steam sterilized and why? 

B. Where is agar derived from? What are the special properties of agar 

that make it well suited as a solidifying agent in culture media? Once 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 6 of 98

For the most current and official copy, check QMiS. 

melted, what temperature should agar be kept at to prevent it from 

solidifying? 

C. In which media and buffer preparations is volume particularly critical? 

D. Why is pH important in media and buffer preparation? 

E. What are some safety concerns when autoclaving? What PPE should 

be utilized when preparing, transporting, and storing media? 

6.1.1.4. References for Media, Reagent, and Supply Preparation 

A. Bacteriological analytical manual (BAM) (current ed.). Center for Food 

Safety and Applied Nutrition, U.S. Food & Drug Administration 

B. AOAC official methods of analysis. (current ed.) Arlington, VA: 

Association of Official Analytical Chemists.

C. U.S. Pharmacopeia/ National Formulary (current ed.) 

D. Difco & BBL Manual: Manual of Microbiological Culture Media (Second 

Edition). 

E. Biosafety in Microbiological and BioMedical Labatoratories (BMBL) 

(current ed.,). Centers for Disease Control and Prevention. 

6.1.2. Safety and Hazardous/Infections Waste 

6.1.2.1. Objective 

A. Introduce the trainee to laboratory safety practices and correctly using 

personal protective equipment (PPE). 

B. Introduce the trainee to the hazards involved in working with pathogens 

and/or their toxins such as Salmonella and Clostridium botulinum toxin. 

C. Develop the trainee’s awareness of procedures for the proper 

segregation and disposal of laboratory waste products. 

D. Identify resources which can assist the employee with the risk 

assessment process, such as state specific laws and requirements. 

E. Be able to utilize the Globally Harmonized System (GHS) for Hazard 

Classification, including being able to recognize standard pictograms for 

hazards, hazard categories, and hazard communication. 

6.1.2.2. Assignment 

A. Read ORA Lab Manual, Volume III, Section 1 for safety issues. 

B. Read Laboratory Chemical Hygiene Plan. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 7 of 98

For the most current and official copy, check QMiS. 

C. Read Laboratory Hazardous Waste Plan. 

D. Read the Biosafety in Microbiological and Biomedical Laboratories 

(BMBL), current edition. 

E. Read the Laboratory Biosafety Plan for local site. 

F. Read the Safety Data Sheets (SDS) for each chemical used in the 

analytical procedure. 

G. Discuss with your trainer or supervisor how etiologic isolates are 

shipped. 

H. Discuss with your trainer, supervisor, and/or industrial hygienist how 

hazardous waste is managed at the local site. 

6.1.2.3. Questions 

A. What are the items of personal protective equipment (PPE), minimally 

needed, in a Biological Safety Level (BSL)-2 biological laboratory? 

B. Describe and give examples of food borne disease microorganisms that 

cause infections, disease, or other health hazards and identify their 

biosafety levels. Describe proper procedures for disposal of used 

media. Describe how to use and operate an autoclave safely. 

C. What is a SDS (Safety Data Sheet)? Where are the SDSs located? 

What information can be found on a chemical reagent hazard label? 

D. Does the state regulate medical waste? What laboratory wastes are 

permitted to enter the sewer? What laboratory wastes are incinerated? 

E. What are the guidelines for handling food microorganisms (mostly 

bacteria or their toxins) in the BMBL? 

F. What are engineering controls? Describe the proper use of these 

engineering controls in a food microbiology laboratory. 

G. What are administrative controls? Describe administrative controls 

designed to minimize the risks of hazardous agent exposure to those 

personnel who are not directly involved with their manipulation. List 

those administrative controls that assist in the maintenance of quality 

control. 

H. What safety equipment is normally found within a microbiological 

laboratory? 

I. What types of laboratory procedures have the potential to generate 

aerosols? How can these procedures be contained? How can the 

generation of aerosols be minimized? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 8 of 98

For the most current and official copy, check QMiS. 

J. What decontamination procedures are in place and when are they 

performed? 

K. Describe how spills are handled. Are the cleanups following a spill 

documented and the cleanup verified? 

L. What kind of signage is to be in place in a microbiological BSL-2 

laboratory? 

M. What are the potential routes of exposure when working with infectious 

organisms? 

N. Why is it necessary for a minimum of two people to be working in the 

laboratory at any given time? 

O. Has enough training been received to perform assigned tasks and has 

this training been documented? 

P. Is the facility designed to prevent infectious organisms from being 

accidentally released to other areas in the building? 

Q. Is there a sharps safety program in place to reduce hazards when 

handling syringes or pipettes or other sharps? 

R. Are animals used to test infectious agents? Identify what program and 

laws address the use of animals in research studies? 

 

6.1.3. Quality Assurance 

6.1.3.1. Objective 

A. Introduce the concept of quality assurance and quality control. 

B. Emphasize the importance of ensuring analytical results and written or 

electronic worksheets are of the highest quality. 

C. Introduce the Quality Management Information System (QMIS). 

D. Discuss the basics and importance of audits. 

6.1.3.2. Assignment 

A. Read the following: 

1) Laboratory Quality Management Plan and International Standard 

ISO/IEC 17025:2017(E). 

2) ORA Lab Manual Volume II, Section 2, and laboratory’s 

corresponding SOPs. 

3) ORA Lab Manual Volume III, Section 3, Records of Results – 

Analyst’s Worksheet 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 9 of 98

For the most current and official copy, check QMiS. 

4) SOP-001064 Microbiological Controls for Food and Feed Product 

Sample Analysis 

B. Trainer will discuss with trainee the various techniques used such as 

positive, negative and system controls, environmental sampling, media 

growth promotion testing, correct media volume, pH and formulation, 

equipment controls, etc. 

C. Trainer should review forms used to report oral and written QA 

evaluations of worksheets. 

D. Trainer should review forms used to report oral and written QA 

evaluations of worksheets. 

E. Trainer should discuss the purpose and importance of internal and 

external audits. 

6.1.3.3. Questions 

 What culture controls are used for an Escherichia coli enumeration 

analysis? For a Staphylococcus aureus analysis? 

 What is the significance of environmental monitoring within an 

accredited microbiology laboratory? How does your laboratory perform 

environmental monitoring? 

 Why are temperatures recorded for incubators, water baths etc.? 

 Why are worksheets reviewed and signed by an analyst who has not 

worked on the sample? 

 Why are validated methods used? 

 What is the purpose of QMIS? What are some of the actions/uses of 

QMIS? 

 What is your laboratory’s current external auditing body? 

6.1.4. Polymerase Chain Reaction (PCR) 

6.1.4.1. Objective 

 Introduce the basic principles and applications of the polymerase chain 

reaction. 

 Introduce the trainee to proper the techniques, procedures, and 

equipment used in PCR analysis. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 10 of 98

For the most current and official copy, check QMiS. 

6.1.4.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of laboratory demonstrations. 

 Read American Society for Microbiology Laboratory Protocols, 

Polymerase Chain Reaction Protocol

 Read Center for Disease Control and Prevention Microbiology Series, 

Basic Molecular Biology Module 4: PCR and Real-Time PCR 

 Practice using a micropipette to dispense small liquid quantities. 

 Practice using proper molecular biology techniques while opening and 

closing small reaction tubes and dispensing reagents. 

 Learn how to calculate correct reagent quantities needed for master mix 

preparation. 

 Trainer will demonstrate how to use a thermal cycler. 

 Trainer will discuss which organisms can be analyzed using PCR. 

6.1.4.3. Questions 

 What are some of the benefits to using PCR to detect microbial 

pathogens in foods? What are some of the potential problems 

associated with using PCR to detect microbial pathogens in foods? 

 What are the three major steps (processes) involved in the PCR cycle? 

 Why would using an enrichment procedure before PCR analysis be 

useful? 

 What components are needed in the PCR reaction mixture and what 

function does each element serve? 

 Why is selection of the primers so important to the success of the 

reaction? 

 Why are there forward and reverse primers? 

 Why does a microbiologist need an excess quantity of primers in the 

reaction mixture? 

 What is real-time PCR? How does it differ from conventional PCR? 

 Why always run a reagent control? 

 List three ways to prevent contamination when performing PCR. 

 In what ways can PCR be inhibited? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 11 of 98

For the most current and official copy, check QMiS. 

6.1.5. Establishment Inspections 

6.1.5.1. Introduction 

Microbiologists are routinely requested to accompany investigators on 

inspection of establishments that produce/store foods or feed. The trainee will 

accompany an investigator and/or an experienced microbiologist. The 

investigator has the basic responsibility for an establishment inspection and 

the microbiologist is a technical advisor in matters pertaining to microbiology, 

but should participate fully in the inspection, noting items for the inspection 

report and final discussion with management. The investigator oversees the 

inspection. 

There are various types of inspections that a microbiologist may be requested 

to assist with such as: seafood HACCP; frozen, chilled, or prepared foods, 

nutmeats; canned food manufacturing; fresh produce processing facility; 

produce farm traceback investigation. Additionally, your role as an analyst on 

inspection may vary from microbiologist subject matter expert reviewing firm 

records and or environmental conditions both inside or outside of the facility, to 

collecting aseptic raw ingredient, in-line, water, environmental swab, or finish 

product samples. 

6.1.5.2. Discussion 

It is the trainer’s primary responsibility to transfer knowledge both practical and 

in theory related to establishment inspections. 

Prior to inspection, the trainee should read and discuss the following with a 

microbiologist experienced in performing establishment inspections to gain a 

fundamental understanding of the various roles you may assume during an 

inspection. 

1) Investigations Operations Manual (IOM), current edition, especially 

Chapter 1 Administration, Subchapter 1.6 -Public Relations, Ethics and 

Conduct; Chapter 4 Sampling, and Chapter 5 Establishment Inspection: 

Food. 

2) ORA Laboratory Manual Volume III, Section 5, Analysts on Inspection 

Prior to inspection, the trainee should talk with the investigator, read the 

previous Establishment Investigations report (EIR), firm history, Safety report 

and read any Compliance Programs or assignment that applies. The trainee 

should also work closely with the senior microbiologist on preparation of 

sample collection materials and on proper protective clothing for the 

establishment. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 12 of 98

For the most current and official copy, check QMiS. 

6.1.5.3. Exercise 

While on the inspection, the trainee should: 

A. Aseptically collect or assist in collection of raw materials, in line 

materials, finished product, water, or environmental swab samples. 

B. Document in a regulatory note book all observations (483 items), 

conversations, questions, equipment, production specific details, 

cleaning procedures, filth and insect control, storage, and all specific 

sample collection information. 

C. Assist with preparing the Sample collection report (CR). 

D. Help prepare and edit the list of observations (Form FD 483) for 

presentation and discussion with firm management. 

E. Contribute to the Establishment Inspection Report and evaluate if the 

report contained enough detailed evidence to support a 

recommendation for legal action. 

F. Evaluate if the analytical results substantiated observations of 

insanitation, OOS (out of specification), or other deficiencies. 

G. Evaluate and discuss if in line sub samples were sufficient to "point out" 

where bacterial contamination could enter and/or proliferate. Evaluate 

and discuss what control measures are in place to control/detect any 

microbiological contamination. 

H. Evaluate the firm’s environmental sampling plan, review the 

methodology and reported findings. 

I. Discuss with the team what aspects of the inspection the trainee would 

like to improve or learn before the next inspection. 

6.2. Basic (Tier 1, i.e. analyses required for analyst level 1 certification) 

The microorganisms listed in this section represent the basic microbiology 

analyses performed in the ORA Food and Feed Microbiology labs. On-the-job 

training and successful mastery of these analyses are key for a new hire 

Microbiologist. This Basic tier of analyses are the minimum set of analyses 

required to become eligible for their New Hire Level 1 Certification. 

A new hire regulatory microbiologist should expect to be issued a minimum of 

two training samples per microorganism type. The second training sample for 

each analyte can be a proficiency or competency sample that demonstrates 

the new hire’s ability to independently work through the primary method with 

minimal assistance. However, the quantity of samples for each analyte may 

be determined by laboratory procedure and or adjusted to the Trainee’s 

individual situation and/or prior experience. Following the successful 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 13 of 98

For the most current and official copy, check QMiS. 

demonstration of competency of each analyte, laboratory management may 

authorize a new hire to begin being assigned regulatory samples as 

determined by individual lab procedure. 

The training sample ideally should be a food or feed sample which is 

representative of some of the common matrices (spices, frozen seafood, 

cheese, produce, etc.) for the lab and for each analysis. Training samples 

shall simulate a regulatory sample in that it helps the new hire understand the 

care and tact that a regulatory microbiologist needs bring to the sample 

analysis. The trainee should have the opportunity to demonstrate appropriate 

chain of custody and aseptic subsampling all throughout the analysis. 

However, the overall assessment criteria should be based upon the successful 

mastery of the analysis. 

Ideally the trainee shall have the opportunity to successfully recover and 

identify the target organism from a blind training sample. Training samples 

shall consist of at least three blind subsamples: one positive or spiked 

subsample (reference SOP-001064 for general guidance on spiking levels and 

spike can occur after sample setup directly into the pre-enrichment); one 

negative subsample spiked with a competitor microorganism (the purpose of 

the negative competitor spike is that it may mimic the target organism 

culturally); and one negative un-spiked subsample. 

6.2.1. Salmonella 

6.2.1.1. Objective 

 Learn about Salmonella, Food Categories, sample compositing, and 

sample analysis. 

 Recover Salmonella by using pre-enrichment and enrichment 

techniques. 

 Screen samples for Salmonella utilizing current rapid methods. 

 Identify Salmonella using biochemical and rapid methods 

 Describe cases and symptoms of Salmonella food borne disease. 

6.2.1.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 14 of 98

For the most current and official copy, check QMiS. 

 Read Bacteriological Analytical Manual (BAM) Chapter 1, Food 

Sampling and Preparation of Sample Homogenate

 Read Bacteriological Analytical Manual (BAM) Chapter 5, Salmonella

 Read AOAC Official Method (2004.03, 2011.03, or other current 

methods utilized in the laboratory) 

 Using BAM/AOAC methods, analyze the minimum number of training 

samples for Salmonella utilizing various sampling techniques and preenrichment broths. 

 The new hire (either through training sample or individual 

demonstration) shall have the opportunity to prepare at least one 15-

sub composite. 

 The trainee shall screen samples using VIDAS AOAC method (or other 

routinely used screening methods) for Salmonella. 

 The trainee (through either training sample or individual demonstration) 

shall inoculate both typical and atypical Salmonella strains onto 

selective agar plates and TSI & LIA slants. 

 The trainer shall ensure that the trainee understands the biochemical 

reactions for both typical and atypical Salmonella and understands 

which can be ruled out and which must continue though confirmatory 

testing. 

 Identify isolates using conventional biochemicals and become familiar 

with the various rapid methods refenced in the BAM and utilized within 

the local laboratory (API 20E, VITEK 2 GN, MICRO ID, VITEK MS, 

LAMP confirmation, and or Real-time PCR). 

 Perform Salmonella serological testing including polyvalent flagellar (H) 

and polyvalent somatic (O) on various serovars as available locally. 

 The trainer should ensure that the trainee understands the basic 

concepts of Salmonella O & H serotyping and how to perform the 

testing according to local practice. 

6.2.1.3. Questions 

 Why are different pre-enrichment media used for different foods? Give 

examples. Why is a pre-enrichment step needed for the recovery of 

Salmonella? 

 Why is more than one type of selective enrichment (RappaportVassilades, selenite cystine and tetrathionate broths) used instead of 

just one? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 15 of 98

For the most current and official copy, check QMiS. 

 Describe sugar reactions in triple sugar iron agar (TSI) tubes. 

 Why use three primary plating media instead of one? 

 Which Salmonella serovars do not typically produce hydrogen sulfide? 

 What is indole? 

 Can we identify all serovars of Salmonella? Give a reason. 

 Describe symptoms and onset time of Salmonella food borne disease. 

 Describe and give examples of foods in Food Category I, II and III. (See 

BAM Chapter 1 Food Sampling) 

 Why is the pH of the enrichment broth adjusted after addition of 

sample? 

 What does the VIDAS assay detect? 

 Why do we need to boil the sample before performing the VIDAS 

assay? 

 Which organism is most likely to cause a VIDAS false positive result? 

6.2.2. Listeria monocytogenes 

6.2.2.1. Objective 

 Evaluate what types of samples may be candidates to be analyzed for 

Listeria monocytogenes. 

 Examine sample matrices and determine appropriate enrichment 

procedures. 

 Choose appropriate methodology involved in the detection of Listeria 

monocytogenes. 

 Differentiate biochemical characteristics between Listeria 

monocytogenes and other Listeria spp. 

 Develop skills in the use of rapid method test kits for the detection and 

differentiation of Listeria species including but not limited to ABI-3500, 

VIDAS, VITEK, RT-PCR, and MICRO-ID. 

 Be able to setup and interpret a MPN and direct plate method for 

Listeria monocytogenes enumeration. 

6.2.2.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 16 of 98

For the most current and official copy, check QMiS. 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) Chapter 1, Food 

Sampling and Preparation of Sample Homogenate

 Read Bacteriological Analytical Manual (BAM) Chapter 10, Listeria. 

 Read Rapid method AOAC Official Methods for Listeria in Foods 

 Using BAM/AOAC methods, analyze the minimum number of training 

samples for Listeria (Basic/Tier 1). Use foods or feed matrices that 

require different subsampling techniques. 

 The new hire (either through training sample or individual 

demonstration) shall have the opportunity to prepare at least one 5-sub 

composite. 

 Screen sample using VIDAS AOAC method (or other routinely used 

screening methods) for Listeria. 

 The new hire (through either training samples or individual 

demonstration) shall inoculate or at a minimum discuss typical Listeria

species including at least 4 different types 

 The trainer shall ensure that the trainee understands the biochemical 

reactions for typical Listeria species and understands which can be 

ruled out and which must continue through confirmatory testing. 

 Identify isolates using conventional biochemicals and become familiar 

with the various rapid methods refenced in the BAM and utilized within 

the local laboratory (VITEK 2, MICRO ID, API listeria, and or Real-time 

PCR). 

 Perform Listeria serological testing including type 1 and 4. 

 The trainer shall ensure that the trainee understands the basic concepts 

and demonstrates how to set up and perform MPN testing of confirmed 

positive samples. 

6.2.2.3. Questions 

 What is unusual about the appearance of the motility of Listeria? 

 At what high and low temperatures will Listeria grow? 

 Describe the CAMP reactions of different Listeria species. What do the 

letters "CAMP" stand for in the CAMP test? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 17 of 98

For the most current and official copy, check QMiS. 

 Why is Rhodococcus equi, and not another Rhodococcus species used 

in the CAMP test? 

 Describe the symptoms and onset time of Listeria food borne disease. 

 Describe the appearance of typical Listeria monocytogenes colonies on 

selective plating media including but not limited to Oxford, Palcam and 

chromogenic agars? 

 Describe how acid production from various carbohydrates (mannitol, 

rhamnose, xylose) typically differs between Listeria monocytogenes and 

Listeria innocua. 

 What role does the addition of selective agents in pre-enrichment and 

enrichment media play? 

 Name two ß-hemolytic Listeria spp and two non ß-hemolytic Listeria 

spp. 

6.2.3. Escherichia coli 

6.2.3.1. Objective 

 Enumerate and identify E. coli in foods. 

 Familiarize the trainee with the different pathogenic strains of E. coli. 

 Understand the MPN technique and how it is calculated. 

6.2.3.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) Chapter 1, Food 

Sampling and Preparation of Sample Homogenate

 Read Bacteriological Analytical Manual (BAM) Chapter 4, E.coli.

 Using BAM/AOAC methods, analyze the minimum number of training 

samples for E.coli (Basic/Tier 1). Use foods or feed matrices that 

require different subsampling techniques. 

 The new hire (either through training sample or individual 

demonstration) shall have the opportunity to prepare at least one 10-

sub sample. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 18 of 98

For the most current and official copy, check QMiS. 

 Screen sample using BAM method and coli complete for E. coli. 

 The new hire (through either training sample or individual sample 

demonstration) shall inoculate media with typical E.coli. 

 The trainer shall ensure that the trainee understands the biochemical 

reactions for both typical and atypical E. coli and understands which can 

be ruled out and which must continue through confirmatory testing. 

 Identify isolates using conventional biochemicals and become familiar 

with the various rapid methods refenced in the BAM and utilized within 

the local laboratory (VITEK 2, VITEK MS, and or IMViC). 

 The trainer shall ensure that the trainee understands the basic concepts 

and demonstrates how to set up MPN testing of confirmed positive 

samples. 

 Analyze or discuss analysis of samples of oysters or clams using the 

"Shellfish Method". 

 Analyze or discuss analysis of finished product shelled walnuts or 

pecans using the "Tree Nut Method”. 

 The trainer will discuss the methods used to determine E. coli 

pathogenicity. 

 Discuss and provide examples for MPN chart exercises. 

 Review and discuss BAM Appendix 2: Most Probable Number from 

Serial Dilutions

6.2.3.3. Questions 

 Describe different types of E. coli that cause food borne disease. List 

methods used for their determination. 

 Is there an acceptable way to minimize foaming when shellfish are 

homogenized? 

 What percent foam is aspirated when pipetting sample from 

homogenate? Would this affect the results? 

 Describe the bottled water definition and how one would initiate 

analysis. 

 Describe how a Gram stain is performed and the meaning of the results. 

 Describe the quadrant streaking technique to obtain isolated colonies. 

 What is the IMViC pattern for E. coli? 

 What does a typical E. coli isolate look like on L-EMB? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 19 of 98

For the most current and official copy, check QMiS. 

 When is it appropriate to use LST-MUG? How does LST-MUG work? 

 What time period is allowable between sample shaking and inoculation 

of tubes? 

6.2.4. Enterohemorrhagic E.coli

6.2.4.1. Objective 

 Isolate and identify E.coli O157:H7 and other non-0157 Shiga-toxin 

producing E. coli (STEC) from food samples. 

 Introduce PCR assay for detection of Shiga-like toxin genes (stx1 and 

stx2) and the WZY gene and applications for food enrichment screening 

and as part of the isolate confirmation. 

 Examine the challenges of recovering E.coli O157:H7 and non-O157 

STEC from foods. 

 Understand the need for complete STEC isolate molecular 

characterization to determine potential health risk. 

 Familiarize the trainee with the different pathogenic strains of E. coli.

6.2.4.2. Assignment 

A. It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

B. Read Bacteriological Analytical Manual (BAM) chapter 4A, 

Diarrheagenic Escherichia Coli online. 

C. Using BAM methods, analyze the minimum number of training samples 

for E.coli, E.coli O157:H7 and if available non-O157 STEC (Basic/Tier 

1). Use foods or feed matrices that require different subsampling 

techniques and pre-enrichment broths. 

6.2.4.3. Questions 

A. Are all STEC also Enterohemorrhagic E.coli (EHEC)? 

B. Do all E.coli strains ferment sorbitol? 

C. Why is tellurite cefixime sorbitol MacConkey agar (TCSMAC) 

particularly useful for the isolation of E. coli O157:H7? 

D. Are all STEC hemolytic? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 20 of 98

For the most current and official copy, check QMiS. 

E. Why is it important to use multiple differential and selective agars to 

recover E. coli O157:H7 and other non-O157 STEC? 

F. Explain why the PCR to confirm the presence of shiga-toxin genes must 

be done as part of the isolate confirmation process even if the PCR was 

used during the initial screening of the food enrichment. 

G. Why is it important to have confirmed STEC isolates further 

characterized for complete virulence profile and serotype by methods 

such as microarray or whole genome sequencing (WGS)? 

6.2.5. Staphylococcus aureus 

6.2.5.1. Objective 

 Analyze foods for S. aureus using both the MPN and direct plating 

techniques. 

 Identify and differentiate coagulase positive versus coagulase negative 

staphylococci. 

 Differentiate biochemical characteristics between Staphylococcus spp. 

 Introduce ELISA techniques for toxin detection. 

6.2.5.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) Chapter 1, Food 

Sampling and Preparation of Sample Homogenate

 Using BAM methods, analyze the minimum number of training samples 

for Staphylococcus aureus (Basic/Tier 1). Use foods or feed matrices 

that require different subsampling techniques. 

 Identify isolates using conventional biochemicals and become familiar 

with BAM specific chapter requirements 

 The trainer shall ensure that the trainee understands the basic concepts 

and demonstrates how to set up MPN testing of confirmed positive 

samples. 

 Read Bacteriological Analytical Manual (BAM) chapter 12, online at: 

Staphylococcus
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 21 of 98

For the most current and official copy, check QMiS. 

 Test or discuss one spiked food for staphylococcal enterotoxin using an 

ELISA based assay (Vidas SET). If a test kit is not found, the trainer is 

responsible for explaining the theory and principals involved. 

6.2.5.3. Questions 

 Describe the symptoms and onset time of staphylococcal food borne 

disease. 

 Describe the difference between intoxication and infection. Which one is 

associated with S. aureus? 

C. Which ingredient/s of Baird Parker medium help injured organisms 

grow? 

D. What are typical observations of coagulase positive Staphylococcus 

aureus when plated on Baird Parker medium? What is indicated by the 

presence or absence of a halo around an isolated colony on Baird 

Parker medium? 

E. Name the staphylococcal enterotoxin types and tell which the most 

common cause of food borne disease is. 

6.2.6. Coliforms 

6.2.6.1. Objective 

 Analyze food and water for coliforms and fecal coliforms 

6.2.6.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) Chapter 1, Food 

Sampling and Preparation of Sample Homogenate

C. Read Bacteriological Analytical Manual (BAM) Chapter 4, Coliforms. 

D. Using BAM method, analyze the minimum number of training samples 

for Coliform and fecal coliforms (Basic/Tier 1). Use foods or feed 

matrices that require different subsampling techniques. 

E. The trainer shall ensure that the trainee understands the biochemical 

reactions for both typical and atypical and understands which can be 

ruled out and which must continue through confirmatory testing. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 22 of 98

For the most current and official copy, check QMiS. 

F. The trainer shall ensure that the trainee understands the basic concepts 

and demonstrates how to set up MPN testing of confirmed positive 

samples. 

G. Use foods prepared for E. coli analysis above for the coliform analysis. 

H. Read and discuss methodology for water test specific to interstate travel 

program using Standard Methods for Water and Wastewater, current 

edition. 

6.2.6.3. Questions 

 Why is sodium thiosulfate added to jars used to collect water samples? 

(See Investigations Operations Manual (IOM), current edition.) 

 Explain the difference between coliforms and fecal coliforms. 

 What organisms are considered coliforms? 

6.2.7. Aerobic Plate Count 

6.2.7.1. Objectives 

 Training should be consistent with methodology utilized in the home 

laboratory. 

 To analyze foods for number of aerobic organisms that grow at 35°C. 

 Demonstrate effective preparation of decimal dilutions from food 

homogenate, milk, cosmetic or product rinse. 

 Apply conventional pour plate technique. 

 Apply spiral plate count (SPLC) method. 

 Distinguish significant figures when calculating APCs or SPLCs. 

 Calculate reporting results for APCs in common and uncommon cases. 

 Utilize proper aseptic technique and quality control principles for 

conventional or SPLC methodology. 

 Apply proper plating procedure. 

 Implement proper sterility controls. 

6.2.7.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 23 of 98

For the most current and official copy, check QMiS. 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) Chapter 1, Food 

Sampling and Preparation of Sample Homogenate

C. Using BAM method, analyze the minimum number of training samples 

for plate count agar (Basic/Tier 1). Use foods or feed matrices that 

require different subsampling techniques. 

D. The trainer shall ensure that the trainee understands the growth 

parameters and common techniques used to establish proper counting 

skill. 

E. The trainer shall ensure that the trainee understands the basic concepts 

and demonstrates how to set up an APC samples. 

F. Read Bacteriological Analytical Manual (BAM) chapter 3, online: APC . 

G. Analyze or discuss at least four foods for APC/g or APC/ml. The trainee 

will report results in correct significant figures. 

6.2.7.3. Questions 

 Name foods that may have natural high counts. Name foods that should 

have low counts. 

 Why are we interested in an APC count? 

 Why do "spreaders" sometimes form when doing an APC? At the airagar interface? At the agar-glass interface? 

D. How long and at what temperature can one "thaw" a frozen food? 

Discuss. 

E. Will there be a change to APC results with repeated freezing and 

thawing of product? 

F. What is the proper procedure for manually mixing dilution blanks? 

G. At what temperature should pour plates be dispensed? 

H. How should the analyst interpret plates with more than 250 colonyforming units (CFUs)? 

I. How should the analyst interpret plates with less than 25 CFUs? 

6.2.8. Yeast and Mold Count 

6.2.8.1. Objective 

 Enumerate colonies of yeasts and molds in foods. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 24 of 98

For the most current and official copy, check QMiS. 

 Introduce dilution and spread plate techniques. 

 Revisit reporting in significant figures. 

6.2.8.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) Chapter 1, Food 

Sampling and Preparation of Sample Homogenate

 Read Bacteriological Analytical Manual (BAM) Chapter 18, Yeast and 

molds. 

D. Using BAM method, analyze at least a minimum number of training 

samples for yeast and mold samples. Use foods or feed matrices that 

require different subsampling techniques. 

E. The trainer shall ensure that the trainee understands the biochemical, 

morphically, or growth differences between yeast and mold. 

F. The trainer shall ensure that the trainee understands the basic concepts 

and demonstrates how to perform Yeast and mold testing of samples. 

G. Read ORA Lab Manual Volume III, Section 4. 

H. Discuss and/or perform tabletop exercise for calculating APC count. 

The trainee will report results in correct significant figures. 

6.2.8.3. Questions 

 Why are we interested in yeast and mold counts? 

 What is the medium of choice for yeast and mold analysis? 

C. What medium is especially useful for analyzing samples containing 

"spreader" molds? 

D. What agent or agents are added to the agar to inhibit bacterial growth? 

E. Why should the plates be left undisturbed until the incubation period is 

complete? 

6.3. Intermediate (Tier 2) 

The microorganisms listed in this section represent the intermediate 

microbiology analyses performed in the ORA Food and Feed Microbiology 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 25 of 98

For the most current and official copy, check QMiS. 

labs. The analyses of some of the microorganisms in this section may need to 

be performed in laboratories that have more experience or specialize in the 

analysis of such organism. Additionally, analysts performing the analyses of 

these microorganisms require advanced analytical technique and ability to 

analyze complex data. At minimum, the analyst should be proficient in 

analyzing microorganisms under the basic microbiology section but does not 

necessarily have to be Level 1 Certified. 

Although proficiency or competency in analyzing microorganisms under this 

section is not required to obtain Level 1 Certification, it is recommended for 

analysts to familiarize themselves with analyzing these complex 

microorganisms and obtain proficiency or competency. On-the-job training 

and successful completion of training for this section should be defined by 

local laboratory procedure. Exercises/Assignments in this tier are intended to 

give introduction to the trainee of the concepts and methodology. Some 

sections may provide additional details for sample training. 

6.3.1. Vibrio 

6.3.1.1. Objective 

 Recover and identify Vibrio cholerae, V. vulnificus and V. 

parahaemolyticus. 

 Introduce polymerase chain reaction (PCR) techniques. 

6.3.1.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) chapter 9, Vibrio

C. Read Bacteriological Analytical Manual (BAM) chapter 28, Detection of 

Enterotoxigenic Vibrio cholerae 

D. It is suggested to examine at least one oyster sample for V. cholerae, V. 

vulnificus, and V. parahaemolyticus. The trainer will spike the sample 

with various Vibrio spp. 

E. It is suggested to examine one food for toxigenic V. cholerae using the 

PCR method. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 26 of 98

For the most current and official copy, check QMiS. 

6.3.1.3. Questions 

 Describe the food borne illness and onset times of the Vibrio spp. that 

were studied. 

 What are the advantages and disadvantages of using a PCR method? 

 Why is NaCl added to media used for Vibrio spp.? 

 Why do some methods enumerate the organism and other methods 

only check for the presence of the organism? 

 What are the characteristics of V. mimicus? What are the similarities 

and differences of this organism compared to V. cholerae? 

 What are the major factors in the pathogenesis of V. cholerae, V. 

parahaemolyticus, V. vulnificus, and V. mimicus? 

6.3.2. Bacillus cereus 

6.3.2.1. Objective 

 Recover and identify B. cereus group organisms from foods. 

 Differentiate biochemical characteristics between various Bacillus spp. 

6.3.2.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

proficiency samples. 

 Read Bacteriological Analytical Manual (BAM) chapter 14, online: B. 

cereus.

C. The trainer should spike dry foods with B. cereus. In addition, the trainer 

should spike foods with Bacillus species that will challenge the student 

to differentiate between the species. 

D. Using BAM method, analyze training samples for B. cereus. Use foods 

or feed matrices that require different subsampling techniques. 

E. The new hire (either through training sample or individual 

demonstration) shall have the opportunity to prepare enough sub 

samples to feel confident about the set-up procedure. 

F. The trainer shall ensure that the trainee understands the biochemical, 

morphological, or growth differences between the different species. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 27 of 98

For the most current and official copy, check QMiS. 

6.3.2.3. Questions 

 Describe the symptoms and onset times for the two types of B. cereus

food borne diseases. What foods have been implicated in each type? 

 Can B. thuringiensis produce diarrheal antigens? Diarrheal disease? 

C. The symptomatic profile of emetic type of food poisoning produced by 

some strains of B. cereus most closely mimics that of Clostridium 

pefringens or Staphylococcus aureus? 

D. The toxins of B. cereus most commonly associated with food poisoning 

are? 

E. Explain the purpose for each ingredient used in MYP agar and how B. 

cereus can be interpreted on MYP agar. 

F. When is the Plate Count Method recommended? When is Most 

Probable Number (MPN) recommended? 

G. When interpreting test results (motility, hemolytic activity, plating 

characteristics, and crystal production), what are typical results for each 

pertaining to B. cereus? 

6.3.3. Campylobacter 

6.3.3.1. Objective 

 Recover and identify Campylobacter jejuni and C. coli from foods and 

water. 

 Introduce microaerobic culturing techniques. 

 Introduce the use of rapid methods for presumptive identification of 

Campylobacter. 

6.3.3.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) chapter 7, online: BAM 

Ch7 Campylobacter 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 28 of 98

For the most current and official copy, check QMiS. 

C. It is suggested to analyze different products such as water, milk/dairy, 

and a poultry product for Campylobacter spp. Trainer will spike samples 

with C. jejuni and C. coli. 

6.3.3.3. Questions 

 Describe the symptoms and onset time of Campylobacter food borne 

disease. 

 Describe methods used to obtain microaerobic conditions. 

C. What are the oxygen-quenching compounds added to Campylobacter

media? 

D. Which biochemical test is used to differentiate between C. jejuni and C. 

coli? 

E. What are the advantages and disadvantages of using rapid methods 

such as the Dryspot Campy Test or Alert for Campylobacter? 

6.3.4. Yersinia enterocolitica 

6.3.4.1. Objective 

 Familiarize the trainee with the public health significance of identifying 

Yersinia species in foods. 

 Familiarize the trainee with growth characteristics of Yersinia 

enterocolitica and Yersinia pseudotuberculosis. 

 Understand how to enrich and identify Yersinia enterocolitica and 

Yersinia pseudotuberculosis in foods. 

6.3.4.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) Chapter 1, Food 

Sampling and Preparation of Sample Homogenate.

 The trainer shall ensure that the trainee understands the biochemical 

reactions for both typical and atypical and understands which can be 

ruled out and which must continue through confirmatory testing. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 29 of 98

For the most current and official copy, check QMiS. 

D. Read Bacteriological Analytical Manual (BAM) chapter 8, Yersinia 

enterocolitica 

E. The trainer will discuss the importance of Yersinia enterocolitica and 

Yersinia pseudotuberculosis as it relates to public health and food 

consumption. 

F. The trainee will enrich food samples for the identification of Yersinia 

enterocolitica and Yersinia pseudotuberculosis. The trainee will identify 

typical Yersinia enterocolitica and Yersinia pseudotuberculosis on 

MacConkey agar plates. 

G. The trainee will identify typical Yersinia enterocolitica and Yersinia 

pseudotuberculosis on Yersinia Selective Agar (YSA) plates (also 

referred to as CIN agar plates). 

H. The trainee will identify typical Yersinia enterocolitica and Yersinia 

pseudotuberculosis on LAIA slants. 

I. The trainee will perform confirmatory and biochemical testing on typical 

Yersinia colonies. 

6.3.4.3. Questions 

 Describe the symptoms and onset time of Yersinia enterocolitica illness. 

 Does Yersinia grow and survive during refrigerated storage? 

 What enrichment broths and selective media are used to culture 

Yersinia enterocolitica? 

 What enrichment broths and selective media are used to culture 

Yersinia pseudotuberculosis? 

E. Describe the biochemical characteristics of Yersinia enterocolitica and 

Yersinia pseudotuberculosis. 

F. What is the relationship of plasmids and Yersinia? 

G. What types of tests are used to determine pathogenicity? 

6.3.5. Clostridium perfringens 

6.3.5.1. Objective 

 Recover and identify C. perfringens from foods. 

 Display anaerobic culture techniques. 

 Differentiate biochemical characteristics between various Clostridium

spp. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 30 of 98

For the most current and official copy, check QMiS. 

D. Effectively utilize Reversed Passive Latex Agglutination (RPLA) test kit 

for the detection of enterotoxin. 

6.3.5.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

Read Bacteriological Analytical Manual (BAM) Chapter 16: Clostridium 

perfringens.

 The trainer should spike food with C. perfringens. The trainee will 

analyze the samples by plate count method using Tryptose-SulfiteCycloserine (TSC) without egg yolk and the alternative plating method 

using TSC with egg yolk. The trainee should also complete all 

presumptive confirmation and completed confirmation testing. If 

possible, the trainee should also be introduced to the RPLA enterotoxin 

test kit. 

6.3.5.3. Questions 

 Describe the symptoms and onset time for C. perfringens food borne 

disease. 

 How would a microbiologist test the organism for toxin production? 

 Tell when a microbiologist would use each enumeration method. 

 How do anaerobe jar commercial systems produce anaerobic 

conditions? How can you tell if it worked? 

 Describe stormy fermentation and how to test C. perfringens for stormy 

fermentation. 

F. Describe key characteristics of C. perfringens including motility, gramreaction, nitrate reduction, and lecithinase activity. 

6.3.6. Clostridium botulinum 

6.3.6.1. Objective 

 Present theory and methodology on recovery of C. botulinum from food. 

 Introduce the method for detection of C. botulinum using the BAM qPCR method 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 31 of 98

For the most current and official copy, check QMiS. 

 Introduce the theoretical concepts of the mouse bioassay. 

 Discuss immunologic and other methods published and/or under 

collaboration to replace the use of animals. 

6.3.6.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

Read Bacteriological Analytical Manual (BAM) Chapter 17: Clostridium 

botulinum 

C. Analyze samples using the BAM Chapter 17 for the detection of 

Clostridium botulinum. 

D. Trainer should spike food with various Clostridium species. 

6.3.6.3. Questions 

 What are the symptoms and onset time of botulism? 

 What is wound botulism and infant botulism? 

C. Name the different types of toxins and which have been implicated in 

human botulism. 

D. Describe how the MLD is calculated. 

E. What is the difference between preformed and formed toxin? 

6.3.7. Alkaline Phosphatase 

6.3.7.1. Objective 

 Discuss the reason we test for alkaline phosphatase in dairy products 

and the reasons a dairy product may contain alkaline phosphatase. 

 Discuss the methods used to test for alkaline phosphatase in dairy 

products and the chemical reactions occurring at various steps in the 

method. 

C. Detect levels of alkaline phosphatase in dairy products using both the 

BAM screening method and the AOAC confirmation method. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 32 of 98

For the most current and official copy, check QMiS. 

6.3.7.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) chapter 27, online: BAM 

CH 27 Alkaline Phosphatase in Cheese 

C. Analyze cheese samples for alkaline phosphatase. The trainer will 

supply samples with known levels of alkaline phosphatase. Samples 

with violative alkaline phosphatase levels will be confirmed with the 

AOAC confirmation method. 

6.3.7.3. Questions 

 Why is the presence of alkaline phosphatase determined in dairy 

products? 

 What is the reason for heating the control blank? 

 Why is a buffer used in the analysis? 

D. In the AOAC confirmation method, why are different buffer and 

precipitant concentrations used for different cheeses? 

E. What parameters need to be controlled during analysis for the phenol to 

be liberated from the disodium phenyl phosphate substrate? 

F. To what compound is the alkaline phosphatase enzyme activity 

proportional? 

G. Describe the reaction that takes place in order for phenol to be 

measured colorimetrically? 

6.3.8. Canned Food and Canned Seam Examination 

6.3.8.1. Objective 

 Learn can classification, progressive decomposition, headspace gas, 

culturing, pH, water activity, direct smear, odor and appearance, etc. 

 Distinguish between an Acidified and Low Acid Canned Food. 

 Familiarize the trainee with other types of hermetically sealed 

containers, i.e., jars & pouches. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 33 of 98

For the most current and official copy, check QMiS. 

D. Discuss 21CFR, Parts 113 and 114 (Low Acid Canned Food and 

Acidified Food Regulations). 

6.3.8.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) Chapter 21A, Chapter 

21B, Chapter 22A, Chapter 22B, Chapter 22C, Chapter 22D, online 

C. Read AOAC Official Method 978.18 Water Activity of Canned 

Vegetables and AOAC Official Method 981.12 pH of Acidified Foods.

D. Read SOP-000620 Water Activity of Foods Using AquaLab 4TE 

E. Read SOP-000621 pH Analysis of Food Products 

F. Read related Compliance Programs 7303.003 Import Acidified & LowAcid Canned Foods and 7303.803(A) Domestic Acidified & Low-Acid 

Canned Foods.

G. Perform a complete canned food analysis, comprising each individual 

test on a representative number of low acid canned foods. 

H. Perform a pH and water activity analysis. 

I. Perform a complete canned seam tear down analysis. 

6.3.8.3. Questions 

 What is a Flipper, Springer, Hard Swell, and Soft Swell? 

 What is flat-sour spoilage? What bacteria can cause this? 

 Does microbial spoilage of a food always result in a swollen can? 

Explain. 

 Why are certain canned food media incubated at 55 degrees C.? 

E. Explain the microbiology and physical attributes of each of the following: 

under processing, leaker spoilage, hydrogen swell, overfill. 

F. Explain the construction of a metal can. Name the components of the 

double seam. 

G. Explain water activity and its significance in an acidified and low acid 

canned food. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 34 of 98

For the most current and official copy, check QMiS. 

6.3.9. Cronobacter spp. (NEW) 

Cronobacter is a gram-negative rod within the family Enterobacteriaceae. 

Reports of Cronobacter infection usually occurs in infants, however clinical 

cases in adults have been described as well. While a reservoir for 

Cronobacter sakazakii is unknown, evidence suggests powdered infant 

formulas as a vehicle for infection. The method for detection and isolation of 

Cronobacter spp. is found in BAM Chapter 29. This method includes a realtime PCR for rapid screening and a cultural method for isolation/confirmation. 

6.3.9.1. Objective 

 Become familiar with Cronobacter spp. and understand their 

significance as a public health concern. 

 Become familiar with FDA validated methods to detect Cronobacter

spp. in food. 

6.3.9.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Consult the following website for more information: BAM Chapter 29. 

Cronobacter

 Perform analysis of infant formula samples for detection, isolation, and 

confirmation of Cronobacter spp. using the appropriate FDA method 

and successfully interpret and report results. 

6.3.9.3. Questions 

 What species belong to genus Cronobacter and which species have 

been involved in human illnesses? 

 What is the morphology of typical colonies on chromogenic agars? 

 Can the method be performed without running the qPCR assay? 

6.3.10. Cosmetic Analysis 

6.3.10.1. Objective 

 Gain theoretical knowledge of cosmetic microbiology. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 35 of 98

For the most current and official copy, check QMiS. 

 Analyze cosmetic products for microorganisms that may cause injury to 

consumers, including pathogenic bacteria and yeast and mold (e.g. 

bacterial contamination of eye cosmetics). 

 Familiarize analysts with the yeast and mold count. 

D. Familiarize analysts with preservative systems employed in multiple use 

eye area cosmetics. 

E. Familiarize analysts with Gram negative, non-fermenter identification. 

F. Familiarize analysts with opportunistic pathogens present in eye area 

cosmetics. 

6.3.10.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read Bacteriological Analytical Manual (BAM) chapter, online: 

Cosmetic 

 Trainer should spike and discuss different matrices and ensure trainee 

should receive spiked subs with Pseudomonas, other Gram-negative 

rods, Gram positive rods and cocci, and yeast. 

6.3.10.3. Questions 

 Describe the sample preparation techniques for liquids and semi 

powders; solids and powders; preparations with petroleum base; 

aerosols of powders and liquids; and aerosols of soaps and other foamy 

liquids. 

 What is the purpose of dilutions and other added ingredients? 

 How do Pseudomonas and Klebsiella differ biochemically? What 

characteristics clearly separate them? 

D. If, biochemically, a culture was indicated to be Pseudomonas but it did 

not produce a fluorescent yellow or blue pigment, would a microbiologist 

still consider the culture as Pseudomonas? Explain. 

E. Which organisms are considered pathogenic in the eye area? On the 

skin? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 36 of 98

For the most current and official copy, check QMiS. 

6.3.11. Nutritional and Dietary Supplements (NEW) 

6.3.11.1. Objective 

 Determine the presence of viable aerobic microorganisms and 

pathogenic microorganisms in pharmaceuticals, from raw materials to 

finished product. 

 Practice aseptic technique when handling isolates. 

 Introduce the use of Official Monographs from the USP: (current 

edition) 

1) 〈2021〉 Microbial Enumeration Test-Nutritional and Dietary 

Supplements 

2) 〈2022〉 Microbiological Procedure for Absence of specified 

Microorganisms- Nutritional and Dietary Supplements 

3) 〈2023〉 Microbiological Attributes of Nonsterile Nutritional and 

Dietary Supplements 

D. Introduce the following concepts: inhibition, inactivating agents, and 

neutralization of inhibiting substances. 

6.3.11.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read USP<2021, 2022 and 2023> (current edition). 

 Analyze or discuss a solid, fluid, water-immiscible fluid, and fluid 

specimen in aerosol form for Total Aerobic Microbial Count, 

Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, 

Escherichia coli, Molds, and Yeast Counts or according to specifications 

of Official Monographs. 

D. Perform preparatory test. 

6.3.11.3. Questions 

 What are the controls used in this experiment? 

 What are some ways to neutralize inhibitory substances? 

 What is Purified Water? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 37 of 98

For the most current and official copy, check QMiS. 

 How does someone determine what microbial test to run on products? 

E. What is the preparatory test? 

6.4. Advanced (Tier 3) 

The analyses and subsequent identification of microorganisms in this section 

may require more advanced training and/or understanding of complex FDA 

methodology. As a result, analyses/organisms in this section are categorized 

as Tier 3 throughout this document. Analyses/organisms that fall under this 

category include, but are not limited to the following: Viruses, Cyclospora, 

Select Agents, and Whole Genome Sequencing. 

Familiarity with Tier 3 analyses/organisms is recommended for new hire 

Microbiologists, however the demonstration of competency in these areas are 

not required to obtain a Level 1 Certification. If an analyst is 

interested/selected to perform any of these advanced topics, competency will 

have to be established prior to working on regulatory analysis in these areas. 

Demonstration and documentation of competency in these areas may be 

determined by the individual laboratory, however the following should be 

included as a minimal requirement: completion of official OTED training course 

(if one is available), comparable on-the-job training by a competent and 

certified trainer, and/or acceptable outcome on a proficiency/audit examination. 

Exercises/Assignments in this tier are intended to give introduction to the 

trainee of the concepts and methodology. Some sections may provide 

additional details for sample training. 

6.4.1. Viruses 

6.4.1.1. Objective 

 Become familiar with viral pathogens capable of causing foodborne 

illness. 

1) Viruses are infectious microorganisms, much smaller than most 

bacteria, which cannot grow or reproduce apart from a living cell. 

They consist of genetic material, either DNA or RNA, encased in 

a protein coat known as the capsid. Several viruses have been 

associated with foodborne illness including Rotaviruses, 

Noroviruses and Hepatitis A. Viruses are difficult to propagate in 

culture but are readily detected using molecular methods such as 

PCR and qPCR. 

2) Human enteric viruses can be transmitted by water, food, 

fomites, and by human contact. They typically have a low 

infectious dose which makes them an immediate public health 

concern. The infectious dose can be a little as one to ten virions 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 38 of 98

For the most current and official copy, check QMiS. 

with a secondary attack rate of 50%. Enteric viruses are the 

leading cause of foodborne infectious diseases in the United 

States and of the 9.4 million annual estimated cases of known 

foodborne illnesses, 58% are attributed to human noroviruses. 

Due to its severity, hepatitis A virus (HAV) is also a concern. 

3) Norovirus (NoV) is a single-stranded, polyadenylated RNA virus 

and can be separated into seven genogroups (GI, GII, GII, GIV, 

GV, GVI, and GVII) based on the sequences of the RNA 

polymerase and capsid regions. Genogroups I, II, and IV are 

known to infect humans, with norovirus GII.4 being the most 

common strain identified. 

4) Hepatitis A is a non-enveloped RNA virus 27 to 32 nm in 

diameter. It has an icosahedral symmetry and belongs to the 

genus Hepatovirus of the Picornaviridae family. Infections with 

HAV can produce effects that range in severity from 

asymptomatic to death from fulminant hepatitis. Infections with 

HAV are typically self-limiting and do not result in chronic liver 

disease, however, outbreaks of HAV require considerable 

amounts of public health resources due to long incubation and 

shedding period after infection. 

B. Become familiar with methods to detect viral food pathogens. 

A. Human noroviruses and wild-type hepatitis A virus detected in 

foodborne outbreaks are non-culturable, therefore detection is based 

primarily upon RT-PCR assays. Advancements in RT-PCR technology 

have allowed the recent development of several RT-qPCR assays for 

the rapid detection and enumeration of norovirus and hepatitis A virus. 

B. Molecular detection methods for RNA viruses depend on converting the 

RNA to cDNA to detect the cDNA by PCR. These molecular detection 

methods must be sensitive enough to detect small amounts of extracted 

RNA. Co-extracted inhibitors from the matrix being tested can often 

cause difficulty in detecting the low levels of virus present in foods. 

PCR methods employ an internal control in the PCR reactions to enable 

the analyst to assess the amount of inhibition is present in the PCR 

reaction. The analysts should be able to assess raw data according to 

the methods and interpret and report results accordingly. 

C. Understand the approaches to detecting viruses in food products. 

1) The most important approach to detecting viruses from foods is 

the concentration step because viruses are usually present in low 

tier amounts. Removing the virus from the food and 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 39 of 98

For the most current and official copy, check QMiS. 

concentrating the virus for molecular level detection is of primary 

importance. The approach is based on the mechanism by which 

virus contamination occurred in the food being tested along with 

the characteristics of the food matrix itself. 

2) Surface contaminated foods are rinsed at a pH higher than the 

isoelectric point (pH at which the virus will absorb to food 

particles) of the target virus. 

3) Internally contaminated food is homogenated prior to lower pH 

adsorption of the virus to the food particle and centrifugation to 

concentrate the adsorbed virus particles. The sample is treated 

at a higher pH to elute the virus from the matrix prior to physical 

separation of the food particles from the elution. Eluted viruses 

are then concentrated at Ultra high speeds to concentrate the 

virus. RNA is then extracted from the sample concentrate and 

tested for virus target RNA. 

4) FDA enteric virus methods rely on successful detection of a 

surrogate virus (Murine Norovirus) which is spiked into the 

sample prior to analysis. Detection of this virus demonstrates 

that the concentration and extraction of virus was successful. 

6.4.1.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Consult the following resources for more information on how these 

approaches are used for various products: 

C. BAM Chapter 26: Concentration, Extraction and Detection of Enteric 

Viruses in Foods 

D. FDA ORA Field Lab Virus Analysis Standard Operating Procedures 

SOP-000578

C. Complete analysis of samples of frozen strawberries, scallops and 

molluscan shellfish using the appropriate FDA method and successfully 

interpret and report results. MNV should be successfully detected, PCR 

data interpretations properly performed, and sample results accurately 

reported. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 40 of 98

For the most current and official copy, check QMiS. 

6.4.1.3. Questions 

 List two ways that viruses differ from bacteria. 

 What is the genetic material found in viruses? 

 What process is required before RNA viruses can be detected by 

qPCR? 

 How are viruses removed and concentrated from fruit, finfish and 

molluscan shellfish? 

 What is the importance of pH in removing viruses from foods and what 

characteristic of the target virus is important in this approach? 

F. How do you determine if the amount of inhibition is too high to be able 

to rely on negative PCR results for a given virus target? 

6.4.2. Cyclospora (NEW) 

Cyclospora cayetanensis is a protozoan parasite responsible for a waterborne 

and foodborne diarrheal illness called cyclosporiasis. C. cayetanensis oocysts 

are spherical measuring 7.5-10 μm in diameter and are unsporulated and not 

infective when freshly passed in stools by infected individuals. Therefore, 

direct fecal-oral transmission cannot occur, and this differentiates Cyclospora

from another important coccidian parasite, Cryptosporidium. After a week or 

longer in the environment at optimal temperatures, C. cayetanensis oocysts 

sporulate and become infective. C. cayetanensis oocysts are not culturable, 

therefore the application of viability assays is impractical. Methods for the 

identification of oocysts in food matrices and water by microscopy are 

available, however these methods are challenging and lack specificity and 

sensitivity to detect low concentrations of oocysts or differentiate between 

morphologically identical species of Cyclospora. As an alternative, methods 

for the detection of C. cayetanensis in fresh produce (BAM Ch. 19b) and 

agricultural water (BAM Ch. 19c) using species-specific qPCR have been 

developed. 

6.4.2.1. Objective 

 Become familiar with Cyclospora cayetanensis and understand its 

significance as a public health concern. 

 Become familiar with FDA validated methods to detect C. cayetanensis

in food and water. 

6.4.2.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 41 of 98

For the most current and official copy, check QMiS. 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Consult the following websites for more information: 

1) BAM Chapter 19b. Detection of Cyclospora cayetanensis in 

Fresh Produce using Real-time PCR 

2) BAM Chapter 19c. Dead-end Ultrafiltration for the Detection of 

Cyclospora cayetanensis from Agricultural Water 

C. Perform analysis of fresh produce samples and/or agricultural water 

samples using the appropriate FDA method. Successfully interpret 

analytical results based on guidance provided in the appropriate BAM 

Chapter. 

6.4.2.3. Questions 

 Explain why methods based on microscopic examination cannot be 

used reliably to detect and characterize C. cayetanensis in food and 

agricultural water. 

 Explain why person to person transmission of C. cayetanensis does not 

occur. 

 Explain the main steps included in the process for molecular 

identification of C. cayetanensis in fresh produce by PCR as described 

in BAM Chapter 19b. 

 Explain the main steps included in the process for molecular 

identification of C. cayetanensis in agricultural water by PCR as 

described in BAM Chapter 19c. 

6.4.3. Whole Genome Sequencing (WGS) (NEW) 

6.4.3.1. Objectives 

 Understand the basic principles and applications of whole genome 

sequencing (WGS) of bacterial isolates. 

 Understand the role and importance of WGS in the regulatory process, 

including what additional information can be gathered, such as 

molecular serological data, virulence markers and phylogenetic 

relatedness to other isolates uploaded to public databases. 

C. Familiarize the trainee with the concept of metadata linked to each 

isolate. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 42 of 98

For the most current and official copy, check QMiS. 

D. Familiarize the trainee with the techniques, procedures and equipment 

used for WGS analyses. 

E. Introduce the use of public GenomeTrakr databases and tools to track 

foodborne pathogens. 

F. Introduce the trainee to the importance of the QA/QC tools available 

and when each tool is required. 

G. Present possible pitfalls and commonly encountered issues during the 

WGS workflow, from DNA extraction to data upload. 

6.4.3.2. Assignment 

 It is the trainer’s primary responsibility to transfer knowledge both 

practical and in theory related to sample analysis. This may be 

accomplished through a series of designated training samples. Once 

the trainer is confident that the trainee can successfully and 

independently perform the analysis, the trainee will be issued a series of 

training samples. 

 Read most recent WGS protocols and SOPs 

 Extract and quantify DNA from isolates. 

 Perform DNA library preparation, pooling and diluting for sequencing 

run. 

E. Operate MiSeq equipment, including starting sequencing run, basic 

washing, and instrument maintenance. 

F. Create sample sheets and program a run utilizing MiSeq Control 

Software. 

G. Understand metadata registration and curation processes and how to 

review isolate status on GenomeTrakr reports and in public databases. 

H. Perform basic QA/QC analysis using MSeq Control Software and 

Sequence Analysis Viewer. 

I. How to review isolate status on GenomeTrakr Reports. 

J. How to review isolate (BioSample) information on NCBI. 

K. How to compare isolate relatedness using phylogenetic trees in NCBI 

Pathogen Detector. 

L. Perform basic QA/QC of sequence data and data transfer procedures. 

6.4.3.3. Questions 

 Describe general steps for WGS analysis of bacterial pathogens. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 43 of 98

For the most current and official copy, check QMiS. 

 What is the main difference between DNA extraction procedures for 

Gram positive and Gram-negative isolates? 

C. Why should index primers be rotated between sequencing runs? 

D. What is the basic sequence coverage requirement for Salmonella, 

Listeria and E. coli? 

E. How often should you wash your sequencer? What should you wash 

your machine with? 

F. Which QA/QC tools should you always run? How do you know when 

you need to utilize additional tools? 

G. What should you do if you suspect an isolate is a result of a 

contamination event involving a laboratory control or another isolate? 

H. What quality metrics should you look for during a sequencing run? 

6.4.4. Select Agents 

6.4.4.1. Objective 

 Introduce the concept that select agents are biological organisms or 

chemical toxins capable of causing great harm or potentially lethal 

disease. In contrast to traditional foodborne pathogens that typically 

cause self-limiting gastrointestinal illness, exposure to microbiological 

select agents can result in serious illness or death. 

 Understand the difference between the different Biosafety Levels, and 

the special requirements for BSL3 laboratories. 

 Become familiar with safety requirements associated with the analysis 

of select agents in foods that are typically analyzed in a BSL3 

laboratory. 

The website: http://www.cdc.gov/od/sap/docs/salist.pdf lists the select 

agents and toxins covered by federal regulation. Some FDA 

laboratories that have BSL-3 registered laboratory space (herein this 

section referred to as “Laboratory facilities”) have received specialized 

training on working with microbiological select agents focusing on those 

microorganisms thought to have the most potential to be used as 

deliberate food contaminants. This specialized training is based on the 

guidelines presented in the current edition of the Biosafety in 

Microbiological and Biomedical Laboratories (BMBL) written by the 

Centers for Disease Control and Prevention and the National Institutes 

of Health and covers several components critical to the safe and secure 

handling of select agents including: 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 44 of 98

For the most current and official copy, check QMiS. 

1) Physical containment - Laboratory facilities must effectively 

contain select agents and the aerosols that could be produced 

during analytical manipulations. This is accomplished through 

design specifications of the facility and use of specialized 

equipment such as Biological Safety Cabinets; sealed centrifuge 

rotors and buckets and transport boxes. 

2) Personal Protective Equipment (PPE) includes closed front 

laboratory gowns or jump suits, double gloves, shoe and hair 

covers and respiratory protection (N95 respirators or PAPRs). 

3) Biosafety practices are designed to contain aerosols and prevent 

release of the select agents. 

4) Methods to Identify Select Agents in various food matrices. 

Recovering select agents from foods can present unique 

challenges depending on the food type. Care must be exercised 

to contain aerosols during processing of the food prior to 

enrichment or biochemical analyses including preparation of 

nucleic acid templates for use in PCR and qPCR analyses. 

D. Become familiar with the federal regulations governing select agents. 

The Centers for Disease Control and Prevention (CDC) regulates the 

possession, use, and transfer of select agents and toxins that pose a 

severe health threat to the public. Laboratories must obtain a Select 

Agent Permit (SAP) from the CDC to handle these organisms. 

Laboratories planning on working with or transferring microorganisms 

that are considered plant or animal pathogens must obtain a permit 

from the USDA Animal and Plant Health Inspection Service (APHIS). 

All laboratories must comply with federal regulations (42 C.F.R. Part 73, 

7 C.F.R. Part 331, and 9 C.F.R. Part 121) regarding biosafety and 

security if they anticipate working with select agents. 

6.4.4.2. Assignment 

Read and discuss the following websites and C.F.R. sections for more 

information on select agents: 

1) www.cdc.gov/od/sap/ 

2) http://www.cdc.gov/biosafety/publications/bmbl5/index.htm 

3) 42 C.F.R. Part 73 

4) 7 C.F.R. Part 331 

5) 9 C.F.R. Part 121 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 45 of 98

For the most current and official copy, check QMiS. 

6.4.4.3. Questions 

 What is a select agent? 

 Name four areas of training necessary before handling select agents. 

 Which agency regulates the use, possession and transfer of select 

agents capable of posing a serious public health threat? 

D. List the PPE required to work with select agents. 

E. What work practices are to be in place when working in a Biological 

Safety Level (BSL)- laboratory? For BSL-3? 

F. Does the laboratory need to register with the CDC when working with 

regulated select agents? 

Document History 

Revision 

# 

Status* 

(D, I, R) Date Author Name and Title Approving Official Name and Title 

1.1 I 10/01/2003 LMEB LMEB 

1.3 R 01/23/2008 LMEB LMEB 

1.4 R 02/06/2012 LMEB LMEB 

1.5 R 03/14/2013 LMEB LMEB 

1.6 R 05/02/2014 LMEB LMEB 

1.7 R 08/25/2014 LMEB LMEB 

02 R 04/27/2021 LMEB LMEB 

03 R REFER TO 

QMIS LMEB LMEB 

* - D: Draft, I: Initial, R: Revision 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 46 of 98

For the most current and official copy, check QMiS. 

Change History 

Revision 

# Change 

1.2 

 Table of Contents – deleted 2.13.3; added 2.15 

 Added Section 2.1 D and 2.3 D.; revised 2.4 B.; revised 2.13, 2.13.1 C., 

and 2.13.2 C. 

1.3 

 Section 2.6 Viruses added 

 Section 2.7 Select Agents added 

 Section 2.17 Document/Change History added Sections 2.6 – 2.14 

renumbered to 2.8 to 2.16 

1.4 

2.1 A. – added sentence to end of paragraph 

2.1 B. – revised first paragraph 

2.1 D. 2. – changed third bullet to serial dilutions 

2.2 B.1. – removed “use” from end of sentence 

2.2 D. – 1. updated; 2. and 3. updated weblink; added 7. 

2.3 B.4. – updated reference 

2.3 C. – revised 2., 8., 9., 10., 11., 14., and 16. 

2.4 B.1 – updated hyperlink 

2.5.1 C. – revised 2., 8., and 13 

2.5.2 B. – revised third sentence in second paragraph 

2.4.2 C. 6. – removed “medium” 

2.5.3 B.1. – revised second sentence 

2.5.3.1. C. 3. – changed media to medium 

2.5.4 C. 3. and 4. – changed agar to medium 

2.5.5 B.2 – removed reference 

2.5.6 C.5. – revised question 

2.5.7 C – 2. analyses changed to analysis; 5. revised question 

2.5.12 B. – defined TSC in last paragraph; removed BAM method 

reference 

2.5 13. B. – second paragraph deleted 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 47 of 98

For the most current and official copy, check QMiS. 

Revision 

# Change 

2.6 A.2. – deleted first sentence 

2.7 – revised third line 

2.7 A.2. – added “USDA” 

2.7 B. – second and fourth link deleted; third link updated 

2.11 A.2. – changed to Acidified and Low Acid Canned Food 

2.11 B. – revised and updated links 

2.11 C.7. – changed to acidified and low acid canned food 

2.13 B.4. – added <71> 

2.13 C. 1 – revised second question 

2.14 A.1. and 2. – revised 

2.14 B – added <61> 

2.15 – updated IOM reference; updated CFR references; added 21 CFR 

111 

2.15 C.3. – added “Firm” 

2.16 Answer Key – revised questions 2.3 1., 2.3 8., 2.3 10., 2.3 11., 2.3 

14., 2.3 16., 2.5.1 2., 

2.5.1 13, 2.5.2 6., 2.5.3.1 3., 2.5.4 3., 2.5.4 4., 2.5.6 5., 2.5.7 2., 2.5.9 1., 

2.8 1., 2.8 2., 2.9 4., 2.11 7., 2.13.1 

1.5 Header – Division of Field Science changed to Office of Regulatory Affairs 

1.6 

 2.4. A. – added “or electronic” 

 2.13. A. 6. – revised 

 2.16 Answer Key – revised questions 1.4.4 and 2.13.3 

1.7 

 2.5.1 – Question 5 –deleted; Question 6 – “species” changed to 

“serovars” & added “typically”; 

 Question 8 – “groups” changed to “serovars” 

 2.16 Appendix – Answer Key – revised questions 2.5.4, 2.5.6, & 2.5.8 

02  Document Reformatted – Outline, numbering and bulleting changed to 

better organize the document 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 48 of 98

For the most current and official copy, check QMiS. 

Revision 

# Change 

 Established new Purpose, Scope, Responsibility, Background, 

References and Procedure sections for this document 

 Realigned the Procedures section into four subsections: Fundamentals, 

Basic (Tier 1), Intermediate (Tier 2), and Advanced (Tier 3); and 

defined each microorganism tier 

 Subsections 2.13 Sterility of Drugs and Medical Devices, and 2.14 

Microbial Limits removed in its entirety; these subsections are covered 

in Volume IV Laboratory Training, Section 15 Drug and Device 

Microbiology 

 Added new subsections for Cronobacter spp., Nutritional and Dietary 

Supplements, Cyclospora, and Whole Genome Sequencing 

 Condensed Establishment Inspections subsections 2.15.1 Frozen, 

Chilled, Prepared Foods; Nutmeats; Shellfish, 2.15.2 Canned Foods 

into a more generalized Establishment Inspection section 

03 

 Fixed formatting issues 

 Updated broken links 

 Updated ORA-LAB.001 to SOP-001064 

 Italicized organism names 

Attachments 

List of Attachments 

Attachment A - Answer Key ........................................................................................ 49
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 49 of 98

For the most current and official copy, check QMiS. 

Attachment A - Answer Key 

FUNDAMENTALS 

Media, Reagent, and Supply Preparation 

A. Which media should not be steam sterilized and why? 

Examples of culture media that are not steam sterilized are Selenite Cysteine Broth, 

Tetrathionate Broth, Bismuth Sulfite and Hektoen Enteric Agars. Autoclaving as well 

as boiling longer than needed destroys the selectivity of the medium. 

B. Where is agar derived from? What are the special properties of agar that make it well 

suited as a solidifying agent in culture media? 

Once melted, what temperature should agar be kept at to prevent it from solidifying? 

Agar is the dried mucilaginous substance extracted from various species of algae. 

The plants are found primarily off the coasts of Japan, China, and southern California. 

Agar is insoluble in cold water but slowly soluble in hot water to give a viscous 

solution. A 1% solution melts at 100°C and sets at 35°C to 50°C to a firm gel. Agar 

should be kept at 55°C to prevent solidifying. Since agar is attacked by relatively few 

bacteria, it is the most satisfactory solidifying agent for the growth and isolation of 

bacterial and fungal species. 

C. In which media and buffer preparations is volume particularly critical? 

Volume of both buffer and media is important to maintain a proper ratio of product to 

media or buffer. Volume is particularly critical in preparing buffer solutions intended 

for use in dilutions for plate counts. Incorrect volume in the buffer blank can result in 

erroneous plate counts. 

D. Why is pH important in media and buffer preparation? 

One of the selective factors in favoring growth of one organism over another is pH. 

For example, media for detection of Vibrio spp., such as TCBS is at pH 8.6. This high 

pH favors growth of the Vibrios while inhibiting competing organisms. Many media 

contain indicators which change color on the acid and basic side. The initial pH of the 

media is neutral with respect to the indicator. Buffer solutions are the initial diluents 

for many analyses and are to maintain a favorable pH environment for the desired 

organism. 

E. What are some safety concerns when autoclaving? What PEE should be worn? 

Autoclave vessels with vented closures only. Do not use crimped seals. Use only 

Pyrex glass vessels and other types of autoclavable materials. Use "liquid cycles." No 

other cycle is safe for liquid sterilization. Follow manufacturer’s instructions for proper 

opening of the door and end of cycle. Do not allow hot bottles to be jolted. This can 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 50 of 98

For the most current and official copy, check QMiS. 

cause hot bottle explosion. Do not move bottles if any boiling or bubbling is present. 

Analysts are required to wear full-length face shields, water impervious aprons and 

rubber gloves whenever removing materials from the autoclave. 

Safety and Hazardous/Infectious Waste 

A. What are the items of personal protective equipment (PPE), minimally required, in a 

Biological Safety Level (BSL)-2 biological laboratory? 

Each person working in a biological laboratory is minimally required to be using safety 

glasses with shields (or goggles, face shield, other splatter guards), a protective lab 

coat, disposable gloves, and protective footwear (closed-toe shoes). 

B. Describe and give examples of food borne disease microorganisms that cause 

infections, disease, or other health hazards and identify their biosafety levels. 

Laboratory access is limited or restricted. Personnel at increased risk for acquiring 

infection, or for whom infection would have serious consequences, are not allowed in 

the laboratory or animal rooms. Laboratory doors are kept closed when experiments 

are in process. Personnel wash their hands after handling microorganisms, when 

removing gloves, and before leaving the laboratory. Soap and disposable towels are 

readily found at sinks in the microbiology laboratories. No eating, drinking, chewing 

gum, smoking, handling medications, handling contact lenses, or applying cosmetics 

is permitted in the laboratory. All food are stored outside of the laboratory. Mouth 

pipetting is prohibited. There is a program for handling and disposing of sharps using 

special sharps' containers. Work surfaces are decontaminated on completion of work 

and at the end of the day. This usually involves a system of various decontamination 

sprays, such as bleach, iodine, quaternium ammonium materials, amphyll, etc. Any 

splashes or spills of viable material are immediately cleaned up with disinfectants that 

are effective against the organism of concern. Contaminated equipment is to be 

decontaminated before it is sent for repair, sent for maintenance, placed in surplus, or 

shipped out of the lab. When placing the equipment in surplus, the equipment is also 

labeled in accordance with the DHHS requirements for property management to show 

that the equipment is clean. All cultures, stocks, and other regulated wastes are 

autoclaved, or otherwise decontaminated, to destroy any viable organisms. All 

materials for autoclaving are placed in red (or otherwise equivalent) bags that are 

closed for transport out of the laboratory. These bags are set in leak-proof totes. Any 

materials to be decontaminated off-site are packaged in accordance with applicable 

local, state, and federal regulations beforehand. When working with organisms with 

more hazardous risk, consideration is given to chemically decontaminating the 

wastes within the laboratory. Insect and rodent control programs are in place. All 

procedures are conducted in a manner to minimize aerosols. Consideration is given 

to the degree of hazard when loops are used in flames, centrifugation, opening 

screw-capped bottles and wet petri dish covers, use of a syringe, needle, and 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 51 of 98

For the most current and official copy, check QMiS. 

septum, streaking plates, pipetting, slide agglutination, etc. Good microbiological 

techniques, substitution of disposable equipment, and use of primary containment 

devices such as biosafety cabinets reduce the risk. Laboratory management 

establishes policies and procedures whereby personnel in the laboratory are informed 

of the potential hazards and meet special entry requirements, such as immunization. 

A biohazard sign is posted at the entrance to any laboratory where etiologic agents 

are used. This label identifies what etiologic agents are used, the biosafety level, any 

required immunizations, the responsible person and their phone number, what PPE is 

worn in the laboratory, and any procedures for exiting the laboratory. Immunizations 

or tests are offered to personnel for the agents handled or potentially present in the 

lab. Baseline blood serum samples may be collected and stored, depending on what 

agents are handled. 

Biosafety procedures are incorporated into individual SOPs, or a Biosafety Manual is 

adopted or prepared for the local laboratory. Personnel are advised of special 

hazards and are instructed to read and follow instructions on practices and 

procedures. Laboratory and support personnel receive training on potential hazards 

associated with the work performed, the precautions to avoid exposure, and exposure 

evaluation procedures. Training is updated annually or when the procedure or policy 

changes. Substitution of glassware for plasticware is recommended. A high degree of 

caution is taken with any procedure using needles and syringes, or any other sharps. 

Sharps should be restricted whenever possible. Only needle-locking syringes or 

disposable syringes should be used for injections, etc. Needles should not be 

manipulated in any manner before disposal; they should be carefully placed into a 

conveniently located puncture-resistant container used for sharps disposal. Nondisposable sharps are to be placed in a hard-walled container for transport to a 

processing area for decontamination, preferably by autoclaving. Syringes that resheathe the needle, needleless systems, and other safety devices are used when 

possible. Never handle broken glassware directly by hand. Use a mechanical means, 

such as brush and dustpan or tongs to pick up broken glass pieces. Dispose of 

broken glass in special containers. Containers of broken glass, sharps, and 

contaminated needles are decontaminated prior to disposal, or are shipped off-site in 

accordance with local, state, or federal regulations. Any cultures, tissues, specimens 

of body fluids, or potentially infectious wastes are placed in a container with a cover 

that prevents leakage during collection, handling, processing, storage, transport, or 

shipping. All spills and accidents that result in overt exposures to infectious materials 

are immediately reported to the Designated Laboratory Official. Medical evaluation, 

surveillance, and treatment are provided as needed and written records are 

maintained in accordance with 29 CFR 1910.1020. Only animals involved with the 

work being performed are permitted in the laboratory. Correct personal protective 

equipment is worn in the laboratory. Disposable gloves are not reused, washed, or 

used when touching “clean” surfaces, such as the telephone, door handles, 

keyboards, etc. Properly maintained safety equipment, including biological safety 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 52 of 98

For the most current and official copy, check QMiS. 

cabinets (Class II) or other containment devices are used whenever procedures with 

a potential for creating infectious aerosols or splashes are conducted, or high 

concentrations or large volumes of infectious agents are used. High concentrations or 

large volumes of infectious material may be centrifuged in the open lab if sealed rotor 

heads or centrifuge safety cups are used and if these rotors or cups are only opened 

inside of the biosafety cabinet. All surfaces inside the laboratory are readily 

accessible for cleaning and can easily be decontaminated. No carpets or rugs are 

used in the laboratory. The biological safety cabinet is located where fluctuations of 

the room supply, and exhaust air do not cause the cabinet to operate outside its 

parameters for containment. The cabinet is located away from doors, from windows 

that can be opened, from heavily traveled laboratory areas, and from other potentially 

disruptive equipment to maintain the biological safety cabinets' air flow parameters for 

containment. Eyewashes are readily found throughout the laboratory and are flushed 

weekly. Outside windows are fitted with screens. Illumination is provided without glare 

or reflections. For BSL-3? All practices in the BSL-2 laboratory are followed in the 

BSL-3 laboratory. All procedures involving the manipulation of infectious materials are 

conducted within biological safety cabinets or other physical containment devices, or 

by personnel wearing correct personal protective clothing and equipment. BSL-3 

laboratory has special engineering and design features, such as a double door 

access zone and sealed penetrations in the facility. The exhaust air from the 

laboratory room is discharged directly to the outdoors. The ventilation to the 

laboratory is balanced to provide directional airflow into the room. Access to the 

laboratory is restricted whenever work is in progress or the BSL-3 organism is 

present, and the recommended Standard Microbiological Practices, Special 

Practices, and Safety Equipment for BSL-3 are rigorously followed. The Designated 

Laboratory Official strictly controls access to the laboratory. Access is restricted to 

only those persons whose presence is needed for program or support purpose. The 

director has the final responsibility for assessing each circumstance and determining 

who may enter or work in the laboratory. The Designated Laboratory Official 

establishes policies and procedures whereby only persons who have been advised of 

the potential biohazard, who meet any identified entry requirements (e.g. 

immunization), and who comply with all entry and exit procedures, enter the 

laboratory or animal rooms. Laboratory and support personnel receive training on the 

potential hazards associated with the work involved, the precautions to prevent 

exposures, and the exposure evaluation procedures. Personnel receive annual 

updates or additional training as needed for procedural changes. The Designated 

Laboratory Official is responsible for ensuring that, before working with organisms at 

BSL-3, all personnel demonstrate proficiency in standard microbiological practices 

and techniques, and in the practices and operations the local laboratory facility. This 

might include prior experience in handling pathogens or cell cultures, or a defined 

training program provided by the director or other competent scientist proficient in 

safe microbiological practices and techniques. All open manipulations involving 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 53 of 98

For the most current and official copy, check QMiS. 

infectious materials are conducted in biological safety cabinets or other physical 

containment devices within the containment module. No work in open vessels is 

conducted on the open bench. Clean up is facilitated by using plastic-backed paper 

toweling on non-perforated work surfaces within biological safety cabinets. Spills of 

infectious materials are decontaminated, contained and cleaned up by 

knowledgeable professional staff, or others properly trained and equipped to work 

with concentrated infectious material. Spill procedures are developed and posted. 

Potentially contaminated equipment is decontaminated before removal from the 

facility for repair or maintenance or packaging for transport, in accordance with 

applicable regulations. Cultures, tissues, specimens of body fluids, or wastes are 

placed in a container that prevents leakage during collection, handling, processing, 

storage, transport, or shipping. All potentially contaminated waste materials (e.g. 

gloves, lab coats, usual waste materials) from laboratories are decontaminated 

before disposal or reuse. Decontamination should occur in the immediate area of the 

BSL-3 unit. Protective laboratory clothing such as solid-front or wrap-around gowns, 

scrub suits, or coveralls are worn by workers when in the laboratory. Protective 

clothing is not worn outside the laboratory. Reusable clothing is decontaminated 

before being laundered. Clothing is changed when overtly contaminated. Gloves are 

worn when handling infectious materials, infected animals, and when handling 

contaminated equipment. Frequent changing of gloves accompanied by hand 

washing is recommended. Disposable gloves are not reused. All manipulations of 

infectious materials, necropsy of infected animals, harvesting of tissues or fluids from 

infected animals or embryonate eggs, etc., are conducted in a Class II or Class III 

biological safety cabinet. When a procedure or process cannot be conducted within a 

biological safety cabinet, then correct combinations of personnel protective 

equipment (e.g., respirators, face shields) and physical containment devices (e.g. 

centrifuge safety cups or sealed rotors) are used. Respiratory and face protections 

are used when in rooms containing infected animals. The laboratory is separated 

from areas that are open to unrestricted traffic flow within the building, and access to 

the laboratory is restricted. Passage through a series of two self-closing doors is the 

basic requirement for entry into the laboratory from access corridors. Doors are 

lockable. Clothes change room may be included in the passageway. Each laboratory 

room contains a sink for hand washing. The sink is hands-free or automatically 

operated and is located near the room exit door. The interior surfaces of walls, floors, 

and ceilings of areas where BSL-3 agents are handled are constructed for easy 

cleaning and decontamination. Seams, if present, are sealed. Walls, ceilings, and 

floors should be smooth, impermeable to liquids and resistant to the chemicals and 

disinfectants normally used in the laboratory. Floors should be monolithic and slipresistant. Consideration should be given to the use of covered floor coverings. 

Penetrations in floors, walls, and ceiling surfaces are sealed or capable of being 

sealed to facilitate decontamination. Openings such as around ducts and the spaces 

between doors and frames are capable of being sealed to facilitate decontamination. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 54 of 98

For the most current and official copy, check QMiS. 

A method for decontaminating all laboratory wastes is found in the facility and utilized, 

preferably within the laboratory (i.e., autoclave, chemical disinfection, incineration, or 

other approved decontamination method). Consideration should be given to means of 

decontaminating equipment. If waste is transported out of the laboratory, it should be 

properly sealed and not transported in public corridors. Consideration is given to safe 

transport of samples potentially contaminated with BSL-3 organisms into the BSL-3 

suite. All such samples should be contained in proper packaging to meet regulatory 

criteria and should not be opened except inside a Class II or III biosafety cabinet. 

A complete list of all regulated organisms appears in 42 CFR 72.3. 

https://www.cdc.gov/selectagent/SelectAgentsandToxinsList.html 

Bacillus anthracis - Biosafety Level 2 practices, containment equipment, and facilities 

are recommended for activities using clinical materials and diagnostic quantities of 

infectious cultures. Animal Biosafety Level 2 practices, containment equipment, and 

facilities are recommended for studies utilizing experimentally infected laboratory 

rodents. Biosafety Level 3 practices, containment equipment, and facilities are 

recommended for work involving production quantities or concentrations of cultures, 

and for activities with a high potential for aerosol production. 

1. Campylobacter (C. jejuni/C. coli, C. fetus subsp. fetus) - Biosafety Level 2 practices, 

containment equipment, and facilities are recommended for activities with cultures or 

potentially infectious clinical materials. 

2. Clostridium botulinum - Biosafety Level 2 practices, containment equipment, and 

facilities are recommended for all activities with materials known to contain or 

potentially to contain the toxin. Additional primary containment and personnel 

precautions, such as those recommended for Biosafety Level 3, are indicated for 

activities with a high potential for aerosol or droplet production, and those involving 

production quantities of toxin. Animal Biosafety Level 2 practices, containment 

equipment, and facilities are recommended for diagnostic studies and titration of toxin. 

3. Escherichia coli (Cytotoxin-producing (VTEC/SLT) organisms) - Biosafety Level 2 

practices, containment equipment, and facilities are recommended for all activities 

utilizing known or potentially infectious clinical materials or cultures. Animal Biosafety 

Level 2 facilities and practices are recommended for activities with experimentally or 

naturally infected animals. 

4. Listeria monocytogenes - Biosafety Level 2 practices, containment equipment, and 

facilities are recommended for activities with clinical specimens and cultures known or 

suspected to contain the agent. Gloves and eye protection should be worn while 

handling infected cultures. Animal Biosafety Level 2 practices, containment equipment 

and facilities are recommended for activities with experimentally or naturally infected 

animals. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 55 of 98

For the most current and official copy, check QMiS. 

5. Salmonella - all serotypes except typhi - Biosafety Level 2 practices, containment 

equipment, and facilities are recommended for activities with clinical materials and 

cultures known to contain or potentially containing the agents. Animal Biosafety Level 

2 practices, containment equipment, and facilities are recommended for activities with 

experimentally or naturally infected animals. typhi - Biosafety Level 2 practices, 

containment equipment, and facilities are recommended for activities with clinical 

materials and cultures known to contain or potentially containing the agents. Animal 

Biosafety Level 2 practices, containment equipment, and facilities are recommended 

for activities with experimentally or naturally infected animals. 

6. Salmonella typhi - Biosafety Level 2 practices, containment equipment, and facilities 

are recommended for all activities utilizing known or potentially infectious clinical 

materials and cultures. Biosafety Level 3 practices and procedures are recommended 

for activities likely to generate aerosols or for activities involving production quantities 

of organisms. 

7. Shigella spp. - Biosafety Level 2 practices, containment equipment, and facilities are 

recommended for all activities utilizing known or potentially infectious clinical materials 

or cultures. Animal Biosafety Level 2 facilities and practices are recommended for 

activities with experimentally or naturally infected animals. 

8. Vibrio enteritis (Vibrio cholerae, V. parahaemolyticus) - Biosafety Level 2 practices, 

containment equipment, and facilities are recommended for activities with cultures or 

potentially infectious clinical materials. Animal Biosafety Level 2 practices, containment 

equipment, and facilities are recommended for activities with naturally or 

experimentally infected animals. 

9. Yersinia pestis - Biosafety Level 2 practices, containment equipment, and facilities are 

recommended for all activities involving the handling of potentially infectious clinical 

materials and cultures. Special care should be taken to avoid the generation of 

aerosols from infectious materials, and during the necropsy of naturally or 

experimentally infected rodents. Gloves should be worn when handling field collected 

or infected laboratory rodents, and when there is the likelihood of direct skin contact 

with infectious materials. Necropsy of rodents is ideally conducted in a biological safety 

cabinet. Additional primary containment and personnel precautions, such as those 

described for Biosafety Level 3, are recommended for activities with high potential for 

droplet or aerosol production, for work with antibiotic-resistant strains, and for activities 

involving production quantities or concentrations of infectious materials. 

10. Bovine spongiform encephalopathy (BSE) cattle BSE prion BoPrPSc - Human prions 

and those propagated in apes and monkeys are manipulated at Biosafety Level 2 or 3, 

depending on the studies being conducted. BSE prions are likewise manipulated at 

Biosafety Level 2 or 3, due to the possibility that BSE prions have been transmitted to 

humans in Great Britain and France. All other animal prions are considered Biosafety 

Level 2 pathogens. Thus, based on the present understanding of prion biology 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 56 of 98

For the most current and official copy, check QMiS. 

described above, once human prions are passaged in mice and mouse PrPSc is 

produced, these prions should be considered Biosafety Level 2 prions, even though 

the human prions are Biosafety Level 3 under most experimental conditions. An 

exception to this statement is in the case of mice expressing human or chimeric 

human/mouse transgenes. These transgenic mice produce human prions when 

infected with human prions and should be treated as Biosafety Level 2 or 3 in accord 

with the guidelines described above. The mechanism of prion spread among sheep 

and goats developing natural scrapie is unknown. CWD, TME, BSE, FSE, and EUE 

are all thought to occur after the consumption of prion-infected foods. 

11. Hantaviruses - Biosafety level 2 practices and procedures are recommended for 

laboratory handling of sera from persons potentially infected with the agents of HPS. 

Potentially infected tissue samples should be handled in BSL-2 facilities following BSL3 practices and procedures. Cell-culture virus propagation should be carried out in a 

BSL-3 facility following BSL-3 practices and procedures. Large-scale growth of the 

virus, including preparing and handling viral concentrates should be performed in BSL4 containment facilities. Experimentally infected rodent species known not to excrete 

the virus can be housed in ABSL-2 facilities using ABSL-2 practices and procedures. 

BSCs and other primary physical containment devices should be used whenever 

procedures with high potential for generating aerosols are conducted. Serum or tissue 

samples from potentially infected rodents should be handled at BSL-2 using BSL-3 

practices and procedures. All work involving inoculation of virus containing samples 

into P. maniculatus or other permissive species should be conducted at ABSL-4. 

C. What is a SDS (Safety Data Sheet)? Where are the SDSs located? What information 

can be found on a chemical reagent hazard label? 

Safety Data Sheets are required documents in OSHA’s Hazard Communication 

standard prepared by manufacturers or distributors of chemicals received by a facility 

to relay information about the hazards of those chemicals, etc. SDSs are located 

where identified for the laboratory and on-line at the web sites of the various 

manufactures or distributors. Chemical labels must relate the manufacturer, his 

address, and phone number in addition to the major hazards of the chemical. The 

health hazards disclosed on the label include, whether the material is a carcinogen, a 

reproductive toxin, a toxic material, a corrosive, damaging to organs, a central 

nervous system depressant, a corrosive, an irritant or sensitizer. Any conditions that 

adversely affect the chemical are also relayed. 

SDSs are located where identified for the laboratory and on-line at the web sites of 

the various manufactures or distributors. Chemical labels must relate the 

manufacturer, his address, and phone number in addition to the major hazards of the 

chemical. The health hazards disclosed on the label include, whether the material is a 

carcinogen, a reproductive toxin, a toxic material, a corrosive, damaging to organs, a 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 57 of 98

For the most current and official copy, check QMiS. 

central nervous system depressant, a corrosive, an irritant or sensitizer. Any 

conditions that adversely affect the chemical are also relayed. 

D. Does the local state regulate medical waste? What laboratory wastes are permitted to 

enter the sewer? What laboratory wastes are to be incinerated? 

EPA no longer regulates medical waste and its destruction, but many states have 

adapted previous EPA requirements and these requirements are still valid. 

E. What are the guidelines for handling food microorganisms (mostly bacteria or their 

toxins) in the BMBL? 

Food organisms are mostly BSL-2 and are handled as any other BSL-2 organism 

described above. 

F. What are engineering controls? 

Controls include use of biological safety cabinets, isolated work areas, use of 

equipment and procedures that reduce manipulation, and employment of ventilation 

and air filtering systems. Describe the proper use of engineering controls in a food 

microbiology laboratory. Properly maintained safety equipment, including biological 

safety cabinets (Class II) or other containment devices are used whenever 

procedures with a potential for creating infectious aerosols or splashes are 

conducted, or high concentrations or large volumes of infectious agents are used. 

Materials that are potentially not contained are managed in the biosafety cabinet. For 

example, infectious material may be centrifuged in the open lab if sealed rotor heads 

or centrifuge safety cups are used and if these rotors or cups are only opened inside 

of the biosafety cabinet. Any operation that potentially generates an aerosol is 

handled in the biosafety cabinet. Plastic backed absorbent paper may be used in the 

cabinet to contain some of the aerosol and reduce an extensive cleanup. The cabinet 

is always decontaminated before and after use with an effective disinfectant. The 

sash is preset to 8-10 inches to minimize any air turbulence in the room that might 

adversely affect the cabinet’s performance. Arm movements in and out of the cabinet 

are slow and minimal to not disrupt the performance. All supplies are placed so they 

are readily found during use of the cabinet. No air grills are blocked during use. All 

contaminated materials are laid in a flat container with disinfectant for later 

decontamination. No flames are used in the cabinet; disposable loops or electronic 

heaters are used for loops. Personnel are dissuaded from walking in front of the 

cabinet during use. A contractor certifies the cabinets annually. 

All contaminated materials are laid in a flat container with disinfectant for later 

decontamination. No flames are used in the cabinet; disposable loops or electronic 

heaters are used for loops. Personnel are dissuaded from walking in front of the 

cabinet during use. A contractor certifies the cabinets annually. 

G. What are administrative controls? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 58 of 98

For the most current and official copy, check QMiS. 

Describe administrative controls designed to minimize the risk of hazardous agent 

exposure to those personnel that are not directly involved with their manipulation. List 

those administrative controls that assist in the maintenance of quality control. 

Cleaning crews should not be allowed in the lab except when scheduled. Only 

authorized personnel should be allowed in the labs and even tighter controls used for 

animal suites. Everything infectious that is transported should be contained. Use of 

plasticware is recommended to reduce the potential for breakage. All wastes are 

autoclaved to ensure nothing infectious enters the sewer system in the building. All 

materials shipped offsite are specially packaged to meet federal requirements. All 

equipment is disinfected before service or surplus. 

H. What safety equipment is found within a microbiological laboratory? 

Eyewashes, sinks, telephones, paper towels, and liquid soap should be readily found 

in every laboratory. Cabinets used for spill supplies, first aid boxes, safety showers, 

fire pull stations, and fire extinguishers should be found in various locations 

throughout the microbiology area. 

I. What types of laboratory procedures have the potential to generate aerosols? How 

can these procedures be contained? How can the generation of aerosols be 

minimized? 

Blending, opening containers of live organisms, using needles and syringes, 

splattering or spilling infectious liquids, injecting needle through septum, opening food 

containers under pressure, splattering droplets of infectious materials in a flame, etc. 

Analysts should use good technique and when needed use containment equipment 

such as biosafety cabinets. 

J. What decontamination procedures are in place and when are they performed? 

Work surfaces are decontaminated on completion of work and at the end of the day. 

This usually involves a system of various decontamination sprays, such as bleach, 

iodine, quaternium ammonium materials, amphyll, etc. Contaminated equipment is 

decontaminated before it is sent for repair, sent for maintenance, placed in surplus, or 

shipped out of the lab. When placing the equipment in surplus, the equipment must 

be also labeled in accordance with the DHHS requirements for property management 

to show that the equipment is clean. 

K. Describe how spills are handled? Are the cleanups following a spill documented and 

the cleanup verified? 

Spills are decontaminated with an effective disinfectant and then cleaned up with 

absorbent pads. Any splashes or spills of viable material are immediately cleaned up 

with disinfectants that are effective against the organism of concern. The material is 

collected as chemical waste if chemicals are also present. If radioactive materials are 

present, the spill is then handled as radioactive waste. The area is monitored to verify 

no hazardous agents are present afterwards by plating swipes of the area. The 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 59 of 98

For the most current and official copy, check QMiS. 

decontaminated microbial spill is handled as a non-hazardous spill if no organisms 

are present. All areas are routinely decontaminated at the end of a project and at the 

end of the day in the microbiological lab. Routine settling plates and air monitoring is 

done to evaluate if there are any infectious issues in the lab. All spills and accidents 

that result in overt exposure to infectious materials should be immediately reported to 

laboratory management. Medical evaluation, surveillance, and treatment are provided 

as needed and written records are maintained in accordance with 29 CFR 1910.1020. 

L. What are the potential routes of exposure when working with infectious organisms? 

The most common potential routes of exposure are inhalation, ingestion, punctures, 

and through the skin absorption and exposure to the mucous membrane of the eye. 

Any cuts in the skin are problematic when working with organisms. 

M. Why is it necessary for a minimum of two people to be working in the laboratory at 

any given time? 

This is a standard policy for laboratories. 

N. Has enough training to perform the assigned tasks been completed and has this 

training been documented? 

All laboratory personnel receive regularly scheduled training on a revolving annual 

basis on safety issues and waste management. This is also part of the training given 

to all new hires. FDA has started the new ORA U on-line training program with webbased training; completion of these modules is documented. 

O. Is the facility designed to prevent infectious organisms from being accidentally 

released to other areas in the building? 

Laboratories have different features. Answers should be reflective of the local lab. 

P. Is there a sharps safety program in place to reduce hazards when handling syringes 

or pipettes or other sharps? 

The answer for all labs should be yes and defined for that lab. 

Q. Are animals used to test infectious agents? Identify what program and laws that 

address the use of animals in research studies? 

Yes. The Animal Welfare Act, IACUC, USDA requirements, and other guidance as 

needed. 

Quality Assurance 

A. What culture controls are used for an Escherichia coli enumeration analysis? For a 

Staphylococcus aureus analysis? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 60 of 98

For the most current and official copy, check QMiS. 

In general, both analyses employ positive and negative cultural controls. The positive 

cultural control assures that conditions at the time of analysis were satisfactory for the 

recovery of the target organism and that the media and biochemical tests give correct 

results. The negative cultural control demonstrates the atypical results for biochemical 

tests and growth characteristics and assures that differential media can give both 

typical and atypical reactions. 

E. coli analysis: 

Biochemically typical E. coli ATCC 25922 is used as a positive cultural control to 

demonstrate the typical growth characteristics and reactions for E. coli: turbidity and 

gas from LST; turbidity, gas, and growth at 45.5o C; gas in BGLB; dark flat colonies on 

EMB agar; indole+, VP-; methyl red+, citrate -. 

Enterobacter aerogenes ATCC 13048 is used as a negative cultural control to 

demonstrate the opposite reactions, so that the analyst is assured that growth 

conditions are satisfactory for E. coli, and our differential media and biochemical 

reagents are working properly for the atypical reaction as well as the typical reaction. 

E. aerogenes does not grow at 45.5o C, produces pink moist colonies on EMB, is 

indole-, VP +, methyl red- , citrate +. 

Staphylococcus aureus analysis: 

Staphylococcus aureus ATCC 6538 is used as a positive cultural control to 

demonstrate the typical growth characteristics and reactions for S. aureus: growth in 

10% salt; black colonies surrounded by an opaque zone on BP agar; coagulase +. 

Staphylococcus epidermidis ATCC14990 is used as a negative cultural control to 

demonstrate atypical reactions on BP, i.e. poor growth with no opaque zone, and 

negative coagulase reaction. 

B. What is the significance of environmental monitoring within an accredited microbiology 

laboratory? How does your laboratory perform environmental monitoring? 

Check Local Guidance for local responses. 

C. Why are temperatures recorded for incubators, water baths etc.? 

Checks of the temperature of instruments such as incubators and water baths are 

needed to maintain confidence in the calibration status of the instrument. For 

instruments that maintain certain temperatures to encourage microbial growth, these 

checks are to be performed frequent. (The ALACC recommends incubators be 

checked twice daily (AM and PM) and water baths be checked daily.) 

Microorganisms only grow at certain temperature ranges, and each has a particular 

optimum temperature. If an incubator or water bath fails to provide that temperature, 

the target pathogen may not be able to recover. It is critical to never exceed the 

temperature range of the organism or it may die off. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 61 of 98

For the most current and official copy, check QMiS. 

D. Why are worksheets reviewed and signed by an analyst who has not worked on the 

sample? 

Worksheets are our “work product” and are used in regulatory actions by the agency. 

They are written by the lead analyst or electronically generated by the web application 

and subsequently reviewed by a check analyst, supervisor, director, compliance 

officer, and in some cases by our legal staff, judges, jury, etc. Not all the readers are 

scientists; therefore, the worksheet should be presented clearly enough for all to 

understand. A poorly presented worksheet will not hold up in court, despite the 

elegant analytical science that may have gone into it. 

It is critical that the worksheet be flawless before it leaves the laboratory. The lead 

analyst or additional analysts may not have detected all the mistakes on the 

worksheet. Therefore, the worksheet is thoroughly reviewed by a peer analyst who did 

not work on the sample, is more objective, and thus is less likely to overlook an error. If 

a peer analyst cannot decipher a worksheet, then a non-scientist has no chance. 

Worksheets are checked for method suitability, accuracy of calculations, accuracy of 

transcribed data, and completeness. 

E. Why use validated methods? 

21CFR2.19 states that it is the policy of FDA to use official methods published in the 

AOAC, whenever possible. These methods have been validated to provide 

documented evidence which provides a high degree of assurance that a procedure 

does what it purports to do (the requirements for an intended use are fulfilled) and is 

reproducible by other laboratories. Use of validated methods ensures consistency 

within the various laboratories in the agency. Use of validated methods ensures that 

our analytical work will hold up in court in defense of our regulatory actions. 

The techniques of validation include using reference standards, comparison of results 

with other methods, interlaboratory comparisons, assessment of factors that influence 

the results, and assessment of uncertainty of the results. Methods are to be 

characterized for detection limit, selectivity, linearity, limit of repeatability and/or 

reproducibility, robustness, and interference from the matrix. Microbiological methods 

are to achieve a low number of false positives, a low number of false negatives, high 

sensitivity, high specificity, and a sensitive limit of detection. 

F. What is the purpose of QMIS? What are some of the actions/uses of QMIS? 

QMIS is the Quality Management Information System. Uses for QMIS include but are 

not limited to SOP/WI/Guidance document edits/creation/archival, Issue 

documentation and tracking, audit finding documentation and tracking, training 

documentation and tracking, storage for documentation, etc. 

G. What is your laboratory’s current external auditing body? 

This response can change every few years so check with local documentation. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 62 of 98

For the most current and official copy, check QMiS. 

Polymerase Chain Reaction (PCR) 

A. What are some of the benefits to using PCR to detect microbial pathogens in food? 

Benefits: It’s fast. Can have results in 1-2 days after sample set-up. It’s sensitive. 

Can detect and amplify very small quantities of target DNA. Problems: Inhibition. 

Inhibition can come from food matrix and will prevent the PCR reaction from working 

correctly. PCR amplifies any target DNA present. DNA can be from living or dead cells. 

An analyst may detect target DNA in a food product, but there may not be viable 

organisms to recover. 

B. What are the three major steps (processes) involved in the PCR cycle? 

Denaturation, annealing (complementary binding of primers), and extension. 

C. Why would using an enrichment procedure before PCR analysis be useful? 

If there are low numbers of injured or stressed bacteria, an enrichment procedure 

would allow the bacteria to recover and grow and improve the chances of detecting the 

organism’s target DNA with PCR. Using a pre-enrichment may also allow enough 

bacteria to grow to facilitate the recovery of viable organisms from the enrichment. 

D. What components are needed in the PCR reaction mixture and what function does each 

element serve? 

Template: provides source of target DNA 

Primers: bind to specific locations on the target DNA 

Nucleotides: provide the building blocks of DNA structure 

Polymerase: extends the DNA strands 

E. Why is selection of the primers so important to the success of the reaction? 

The primers will bind on either side of the target DNA. Where they bind will determine 

what portions of the DNA strand are amplified and how large the PCR product will be. 

F. Why are there forward and reverse primers? 

Primers are short DNA sequences complementary to the single-stranded DNA 

templates from which the polymerase begins replicating. PCR uses forward and 

reverse primers that bind to opposite strands of DNA, flanking the region that is to be 

amplified. DNA synthesis at one primer is directed toward the other, resulting in the 

replication of the desired sequence. 

G. Why does a microbiologist need an excess quantity of primers in the reaction mixture? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 63 of 98

For the most current and official copy, check QMiS. 

To ensure the denatured DNA strands anneal to the primers, not to each other. The 

quantity of primers in the reaction also partly determines how many times the DNA can 

be amplified. 

H. What is real-time PCR? How does it differ from conventional PCR? 

Real time PCR (ex. TaqMan) allows for DNA amplification and measurement to occur 

within the same reaction tube. The machine will measure the increasing fluorescence 

intensity that occurs as increasing amounts of target DNA are amplified. This 

eliminates the step of running the PCR reaction product on a gel to determine if target 

DNA has been amplified. 

More detailed description of what happens during real-time PCR... 

A probe is designed to anneal to the target sequence between the traditional forward 

and reverse primers. The probe is labeled at the 5' end with a reporter fluorochrome 

(usually 6carboxyfluorescein, 6-FAM) and a quencher fluorochrome (6-carboxytetramethylrhodamine, TAMRA) added at any T position or at the 3' end. If both 

fluorochromes are on the probe, the quencher molecule stops all fluorescence by the 

reporter. However, as TAQ polymerase extends the primer, the intrinsic nuclease 

activity of TAQ degrades the probe, releasing the reporter fluorochrome. Thus, the 

amount of fluorescence released during the amplification cycle is proportional to the 

amount of product generated in each cycle. An optical fiber inserted through a lens is 

positioned over each well and laser light is directed through the fiber to excite the 

fluorochrome in the PCR solution. Emissions are sent via the fiber to the CCD camera 

where they are analyzed by the software's algorithms. Collected data is subsequently 

sent to the computer. The software calculates the threshold cycle (CT) for each 

reaction with which there is a linear relationship to the amount of starting DNA. 

I. Why always run a reagent control? 

“DNA free” reagent control should always be run to show that there was no DNA 

present in the reagent to contaminate the PCR reaction. 

J. List three ways to prevent contamination when performing PCR. 

 Prepare PCR solutions in an area different from the amplification area. Use pipettes 

and supplies that are dedicated for PCR use only. 

 Use gloves. 

 Aliquot stock solutions and prepared solutions into small quantities and small 

containers to avoid contamination of a large volume of solution. 

 Avoid spilling and splashing of reagents. Use filtered pipette tips. 

K. In what ways can PCR be inhibited? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 64 of 98

For the most current and official copy, check QMiS. 

PCR inhibitors can interfere with the cell lysis needed for extraction of DNA, interfere 

by nucleic acid degradation or capture, and can inhibit polymerase activity of 

amplification of target DNA. Potential PCR inhibitors include components of body fluids 

(e.g. hemoglobin, urea, and heparin), food constituents (organic and phenolic 

compounds, glycogen, and fats), environmental compounds (phenolic compounds, 

humic acids, and heavy metals), constituents of bacterial cells, non-target DNA, pollen, 

glove powder, laboratory plastic ware, and cellulose. 

Reference: Wilson, I. G. 1997. Minireview Inhibition and Facilitation of Nucleic Acid 

Amplification, Applied and Environmental Microbiology Vol. 60, No. 10 p. 3741-3751. 

Establishment Inspections 

NO QUESTIONS 

BASIC (TIER 1) ORGANISMS

Salmonella spp. 

A. Why are different pre-enrichment media used for different foods? Give examples. Why is 

a pre-enrichment step needed for the recovery of Salmonella? 

Different food matrices inherently involve different intrinsic growth factors. Preenrichment in non-selective mediums takes into account the different intrinsic 

parameters of foods that affect microbial growth thereby, allowing for cell repair, 

diluting toxic or inhibitory substances and providing nutritional requirements for growth. 

The pre-enrichment procedure was devised for use with specified food products to 

promote the growth of injured cells of Salmonella. This step favors the recovery of 

microorganisms from the injured state in which some are believed to exist in many 

foods. A pre-enrichment step is needed because Salmonella organisms may be 

present in small numbers and/or in competition with microflora. Salmonella organisms 

may also be injured in food-processing procedures, which include exposure to low 

temperatures, sub marginal heat, drying, radiation, preservatives, and sanitizers. When 

attempting to initiate growth of inactive bacteria, conditions for their growth such as 

favorable temperature and pH and the nutritional requirements should be maintained 

during analysis. 

Example. Lactose broth – egg containing products, crustaceans, cheese, fresh frozen 

dried fruits and vegetables; T-soy broth – spices; Nonfat Dry Milk (NFDM) – candy; BG 

water – dry milk 

By using a pre-enrichment such as Lactose Broth, other organisms will utilize the 

lactose resulting in a lower pH. This lower pH serves to hold in check the other 

competing organisms which may be present in greater numbers. The lower pH does 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 65 of 98

For the most current and official copy, check QMiS. 

not appear to be sufficient to affect the growth or to be lethal for Salmonella recovery. 

Other pre-enrichment broths have been shown to increase the recovery of Salmonella

in certain products (e.g. TSB for spices, NFDM for chocolate). 

B. Why is more than one type of selective enrichment (Rappaport-Vassilades, selenite 

cystine and tetrathionate broths) used instead of just one? 

More than one type of enrichment (RV, TT, and SC) is used instead of just one to 

increase the chances of recovery. These different enrichment broths showed different 

recoveries depending upon the food matrix and the different serotypes involved. Some 

serotypes may be more sensitive to one medium than another, so more than one 

enrichment broth is used to recover as many serotypes as possible. 

Rappaport-Vassiliadis: This medium selectively enriches for Salmonellae because 

bacteria, including intestinal bacteria, are high resistant to or inhibited by malachite 

green, high osmotic pressure and/low pH. It is used widely for analyzing milk and milk 

products, raw flesh foods, highly contaminated foods, and animal feeds. 

Selenite Cystine: As a selective enrichment medium, Selenite Cystine broth allows the 

proliferation of Salmonella while inhibiting the growth of competing non-Salmonella

bacteria. Selenite broth favors the growth of Salmonella while reducing growth of fecal 

coliforms and enterococci. Sodium Acid Selenite inhibits gram-positive bacteria and 

most enteric gram-negative bacteria except Salmonella. 

Tetrathionate: This selective enrichment is used for the cultivation of Salmonella

species that may be present in small numbers. This medium inhibits or kills the 

coliform organisms and permits the typhoid and paratyphoid bacilli to grow almost 

unrestrictedly. 

C. Describe sugar reactions in triple sugar iron agar (TSI) tubes. 

Lactose and sucrose are present at 1%, while glucose is present at only 0.1%. 

Salmonella ferment glucose but not lactose or sucrose. Phenol red, the pH indicator in 

TSI, is red under alkaline conditions and yellow under acidic conditions. At first, the 

slant and butt turns yellow due to glucose fermentation (acidic). Since the lactose and 

sucrose cannot be utilized, when the glucose is used up, the Salmonella depends on 

the aerobic de-amination of the amino acids in the medium (alkaline). The slant reverts 

to red because of the alkaline aerobic reaction, while the butt remains yellow due to 

the acidic glucose (anaerobic) fermentation. 

An alkaline slant-acid butt (red/yellow) indicate fermentation of dextrose only. 

An acid slant-acid butt (yellow/yellow) indicates fermentation of dextrose, lactose, or 

sucrose. (Sucrose is added to TSI to eliminate some sucrose-fermenting non-lactose 

fermenters such as Proteus and Citrobacter spp.) 

An alkaline slant-alkaline butt (red/red) indicates that neither dextrose nor lactose was 

fermented (non-fermenter). 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 66 of 98

For the most current and official copy, check QMiS. 

D. Why use three primary plating media instead of one? 

The use of 2 or more selective plating media is recommended to facilitate the recovery 

of strains of Salmonella that may be inhibited by a particular plating medium. Some 

Salmonella serotypes grow poorly or fail to grow on certain selective agars (e.g. S. 

typhi is inhibited by BG agar). Selective plating is the procedure requiring the use of 

solid selective media (BS, HE, XLD) that restrict growth of bacteria other than 

Salmonella and provide visual recognition of pure, discrete colonies suspected to be 

Salmonella. 

Bismuth Sulfite (BS) – A highly selective medium used for the isolation of S. typhi and 

other Salmonella. Gram positive bacteria and members of the coliform group are 

inhibited. The inhibitory reaction to gram positive and coliform organisms permits the 

use of much greater inoculum. The use of larger inoculum increases the possibility of 

recovering pathogens especially if present in low numbers. 

Hektoen Enteric (H E) – A selective, differential medium for the isolation and 

differentiation of Salmonella from gram negative pathogens (The lactose positive 

colonies are differentiated from the lactose negative colonies) while at the same time 

ensuring inhibition of gram-positive microorganisms. 

Xylose Lysine Deoxychocolate (XLD) – A selective, differential plating medium. Inhibits 

the growth of gram-positive organisms. Salmonella are differentiated from the other 

xylose fermenting enteric organisms with the addition of lysine. Salmonella organisms 

decarboxylate lysine causing a reversion to alkaline conditions. Alkaline reversion by 

other lysine positive organisms is prevented by excess acid production from 

fermentation of lactose and sucrose. 

E. Which Salmonella serovars do not typically produce hydrogen sulfide? 

S. ser. Paratyphi and S. ser. choleraesuis. Other serotypes may also have mutations 

which produce hydrogen sulfide negative strains. 

F. What is indole? 

Indole is a component of the amino acid tryptophan. Some bacteria can break down 

tryptophan for nutritional needs using the enzyme tryptophanase. When tryptophan is 

broken down, the presence of indole can be detected using Kovacs' reagent. Kovac's 

reagent, which is yellow, reacts with indole and produces a red color on the surface of 

the test tube. Salmonella cultures give a negative test (lack of deep red color at 

surface of broth). 

Indole test: 0.2 - 0.3ml of Kovacs reagent is added to a 5 ml aliquot of a 24-hour 

tryptophan broth culture. 

G. Can all serovars of Salmonella be identified with antisera? Give reason. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 67 of 98

For the most current and official copy, check QMiS. 

No. Salmonella is not identified with antisera alone. Identification calls for answers 

from biochemical reactions as well as serological reactions. When all pieces are put 

together, one has an identification. An example is the two non-motile species (S. 

bioser Gallinarum, and S. bioser Pullorum). Both contain the same O antigens, but no 

H antigens. They are to be identified biochemically. 

H. Describe symptoms and onset time of Salmonella food borne disease. 

Acute symptoms - Nausea, vomiting, abdominal cramps, diarrhea, fever, and 

headache. Chronic consequences -- arthritic symptoms may follow 3-4 weeks after 

onset of acute symptoms. 

Onset time – 6 to 72 hours. 

Infective dose - As few as 15-20 cells; depends upon age and health of host, and 

strain differences among the members of the genus. 

Duration of symptoms - Acute symptoms may last for 1 to 2 days or may be prolonged, 

again depending on host factors, ingested dose, and strain characteristics. Cause of 

disease - Penetration and passage of Salmonella organisms from gut lumen into 

epithelium of small intestine where inflammation occurs; there is evidence that an 

enterotoxin may be produced, perhaps within the enterocyte. 

I. Describe and give examples of foods in Food Category I, II and III. (See BAM chapter 1 

Food Sampling.) 

Food Category I. - Foods that would not normally be subjected to a process lethal to 

Salmonella between the time of sampling and consumption and are intended for 

consumption by the aged, the infirm, and infants. 

Food Category II. - (Numbers in chart represent industry codes) - Foods that would 

not normally be subjected to a process lethal to Salmonella between the time of 

sampling and consumption. 

2 Milled grain products not cooked before consumption (bran and wheat germ) 

3 Bread, rolls, buns, sugared breads, crackers, custard- and cream-filled sweet goods, 

and icings 

5 Breakfast cereals and other ready-to-eat breakfast foods 

7 Pretzels, chips, and other snack foods 

9 

Butter and butter products, pasteurized milk and raw fluid milk and fluid milk 

products for direct consumption, pasteurized and unpasteurized concentrated liquid 

milk products for direct consumption, dried milk and dried milk products for direct 

consumption, casein, sodium caseinate, and whey 

12 Cheese and cheese products 

13 Ice cream from pasteurized milk and related products that have been pasteurized, 

raw ice cream mix and related unpasteurized products for direct consumption 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 68 of 98

For the most current and official copy, check QMiS. 

14 Pasteurized and unpasteurized imitation dairy products for direct consumption 

15 Pasteurized eggs and egg products from pasteurized eggs, unpasteurized eggs and 

egg products from unpasteurized eggs for consumption without further cooking 

16 

Canned and cured fish, vertebrates, and other fish products; fresh and frozen raw 

shellfish and crustacean products for direct consumption; smoked fish, shellfish, and 

crustaceans for direct consumption 

17 Meat and meat products, poultry and poultry products, and gelatin (flavored and 

unflavored bulk) 

20-

22 

Fresh, frozen, and canned fruits and juices, concentrates, and nectars; dried fruits 

for direct consumption; jams, jellies, preserves, and butters 

23 Nuts, nut products, edible seeds, and edible seed products for direct consumption 

24 Vegetable juices, vegetable sprouts, and vegetables normally eaten raw 

26 Oils consumed directly without further processing; oleomargarine 

27 Dressings and condiments (including mayonnaise), salad dressing, and vinegar 

28 Spices, flavors, and extracts 

29 Soft drinks and water 

30 Beverage bases 

31 Coffee and tea 

33 Candy (with and without chocolate; with and without nuts) and chewing gum 

34 Chocolate and cocoa products 

35 Pudding mixes not cooked before consumption, and gelatin products 

36 Syrups, sugars, and honey 

37 Ready-to-eat sandwiches, stews, gravies, and sauces 

Food Category III. - Foods that would normally be subjected to a process lethal to 

Salmonella between the time of sampling and consumption. 

2 Whole grain, milled grain products that are cooked before consumption (corn meal 

and all types of flour), and starch products for human use 

3 Prepared dry mixes for cakes, cookies, breads, and rolls 

4 Macaroni and noodle products 

16 

Fresh and frozen fish; vertebrates (except those eaten raw); fresh and frozen 

shellfish and crustaceans (except raw shellfish and crustaceans for direct 

consumption); other aquatic animals (including frog legs, marine snails, and squid) 

18 Vegetable protein products (simulated meats) normally cooked before consumption 

24 Fresh vegetables, frozen vegetables, dried vegetables, cured and processed 

vegetable products normally cooked before consumption 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 69 of 98

For the most current and official copy, check QMiS. 

26 Vegetable oils, oil stock, and vegetable shortening 

35 Dry dessert mixes, pudding mixes, and rennet products that are cooked before 

consumption 

J. Why is the pH of the enrichment broth adjusted after addition of sample? 

The pH for optimum growth is around neutrality, with values above 9.0 and below 4.0 

being bactericidal. For best growth, the Salmonella require a pH between 6.6 and 8.2. 

K. What does the VIDAS assay detect? 

VIDAS Salmonella is an enzyme immunoassay for the detection of Salmonella

antigens using the ELFA (Enzyme Linked Fluorescent Assay) method. Vidas assay 

detects both motile and non-motile Salmonella. 

The SPR (Solid Phase Receptacle) is coated with anti-Salmonella antibodies. 

Salmonella antigens present in the sample will be bound. Unbound sample 

components are washed away. Antibodies conjugated with alkaline phosphatase are 

cycled in and out of the SPR and will bind to the Salmonella antigen bound by the 

SPR. A final wash step removes unbound conjugate. A fluorescent substrate, 4-

methyl-umbelliferylphosphate, is introduced in the SPR. This enzyme will catalyze the 

conversion of the substrate to the fluorescent product, 4methyl-umbelliferone. The 

intensity of the fluorescence is measured by the optical scanner in the VIDAS. 

L. Why does the sample need to be boiled before performing the VIDAS assay? 

The culture needs to bed boiled before performing the VIDAS assay to kill the 

organism. Otherwise, the VIDAS would become contaminated. The boiling procedure 

disrupts the cell wall thereby, making accessible the antigen for the antigen-antibody 

reaction. 

M. Which organism is most likely to cause a VIDAS false positive result? 

The organism most likely to cause a false positive result is Citrobacter. Citrobacter may 

contain common O, H, and Vi antigens. The pH for optimum growth is around 

neutrality, with values above 9.0 and below 4.0 being bactericidal. For best growth, the 

Salmonellae require a pH between 6.6 and 8.2. 

 

Listeria monocytogenes 

A. What is unusual about the appearance of the motility of Listeria? 

 A characteristic motility of Listeria is described as tumbling. 

B. At what high temperatures and low temperatures will Listeria grow? 

Listeria can grow between 2.5 and 44°C 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 70 of 98

For the most current and official copy, check QMiS. 

C. Describe the CAMP reactions of different Listeria species. What do the letters “CAMP 

stand for in the CAMP test? 

“CAMP” stands for Christie-Atkins-Munch-Peterson. 

Hemolytic Interaction 

Species Staphylococcus aureus Rhodococcus equi

L. monocytogenes + - (a)

L. ivanovii - + 

L. innocua - - 

L. welshimeri - - 

L. seeligeri + - 

a Some strains are weakly R+, especially after 48hr incubation, but the R+ 

reaction is much less pronounced than that of L. ivanovii 

D. Why is Rhodococcus equi, and not other Rhodococcus species used in the CAMP 

test? 

There is a synergistic hemolytic effect based on the diffusion of "R. equi factors," 

phospholipase C and cholesterol oxidase which help enhance hemolysis of some 

species of Listeria when performing the camp test. 

E. Describe the symptoms and onset time of Listeria foodborne disease. 

Flu-like illness with fever, myalgia or headache and diarrhea. Onset can be days to 

weeks except for gastroenteritis which has a rapid onset (hours). 

F. Describe they appearance of typical Listeria monocytogenes colonies on selective 

plating media including but not limited to Oxford, Palcam, and BCM. 

Listeria monocytogenes has a similar appearance on Oxford and Palcam with black 

pin-point colonies with a black halo. L. monocytogenes has a blue colony on BCM. 

G. Describe how acid production from various carbohydrates (mannitol, rhamnose, 

xylose) typically differs between Listeria monocytogenes and Listeria innocua. 

Acid Production 

Species Mannitol Rhamnose Xylose 

L. monocytogenes - + -

L. innocua - V(b) - b V, variable 

H. What role does the addition of selective agents in pre-enrichment and enrichment 

media play? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 71 of 98

For the most current and official copy, check QMiS. 

To increase the number of Listeria by minimizing background microflora. 

I. Name two beta-hemolytic and two non beta-hemolytic Listeria species. 

Beta-Hemolytic: L. monocytogenes and L. ivanovii 

Non Beta-Hemolytic: L. innocua and L. welshimeri 

Escherichia coli

A. Describe different types of E. coli that cause food borne disease. List methods used for 

their determination. 

 Enterotoxigenic E. coli (ETEC)-gastroenteritis, traveler's diarrhea 

 ETEC strains produce two types of toxins: heat-labile toxin (LT) and heat-stable 

toxin (ST). LT can be detected by the Y-l tissue culture test, and ST can be 

detected by the infant mouse test. These toxins also can be detected by ELISA, 

and genes coding for them can be detected by gene probes. 

 Enteropathogenic E. coli (EPEC)-infant diarrhea 

Although there are no definitive tests for EPEC strains, some methods, which 

are still experimental, are found in the literature or from communications with 

researchers. Confirmation of a putative EPEC strain involves serogrouping and 

serotyping and consideration of case symptomology. Further complexity 

concerns the fact that some EPEC strains behave like EHEC strains. 

 Enterohemorrhagic E. coli (EHEC)-hemorrhagic colitis 

The toxins produced by EHEC are detected by tissue culture assays. However, 

DNA probe and polymerase chain reaction assays have also been developed to 

detect the presence of SLT gene in HEC isolates. 

 Enteroinvasive E. coli-(EIEC)-bacillary dysentery 

A tissue culture (HeLa cell) test is found in the BAM to screen isolates for 

invasive potential before confirming invasiveness by the Sereny test. An in vitro 

staining technique using acridine orange to stain for intracellular (invasive) 

bacteria in HeLa cell monolayers is also an effective assay to determine 

invasiveness of pathogenic E. coli.

 Enteroadherent E. coli (EAEC)-newly added category 

Is not fully characterized in the BAM and so no methodology is not found there. 

B. Is there an acceptable way to minimize foaming when shellfish are homogenized? 

Yes, limit the blending to 60-90 seconds. Excessive grinding can cause overheating 

which leads to the foaming. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 72 of 98

For the most current and official copy, check QMiS. 

C. What percent foam is aspirated when pipetting sample from homogenate? Would this 

affect the results? 

Less than 5% is aspirated. The BAM specifies that the solution should be allowed to 

settle. The top is not to be pipetted. The effect of the foam is that it destroys any 

protein produced by the bacteria. This is important in characterization that involves the 

protein. 

D. Describe the bottled water definition how would one initiate analysis. 

In addition to “bottled water” or “drinking water”, in 21 CFR Part 103 FDA also defines 

various types of bottled water that meet certain criteria. These identities include 

"artesian or artesian well water", "ground water", mineral water", "purified or 

demineralized water", "sparkling bottled water", "spring water" and "well water". Under 

the current bottled water quality standard, FDA has established a microbiological 

quality requirement that is based on coliform detection levels. These levels may be 

obtained by membrane filtration (MF) or by 10-tube MPN analysis of ten 10-mL 

analytical units. 

E. Describe how a Gram stain is performed and the meaning of the results. 

Gram Stain Procedure 

 Add crystal violet stain over the fixed culture. Let stand for 10 to 60 seconds; for 

thinly prepared slides, it is usually acceptable to pour the stain on and off 

immediately. Pour off the stain and gently rinse the excess stain with a stream of 

water. Note that the objective of this step is to wash off the stain, not the fixed 

culture. 

 Add the iodine solution on the smear, enough to cover the fixed culture. Let stand 

for 10 to 60 seconds. Pour off the iodine solution and rinse the slide with running 

water. 

 Shake off the excess water from the surface. 

 Add a few drops of decolorizer so the solution trickles down the slide. Rinse it off 

with water after 5 seconds. The exact time to stop is when the solvent is no longer 

colored as it flows over the slide. Further delay will cause excess decolorization in 

the gram-positive cells, and the purpose of staining will be defeated. 

 Counterstain with basic fuchsin solution for 40 to 60 seconds. Wash off the solution 

with water. Blot with bibulous paper to remove the excess water. Alternatively, the 

slide may be shaken to remove most of the water and air-dried. 

 Meaning of the results - If the stained culture retains the primary dye (the purplish 

crystal violet dye, then the culture is gram positive. However, if the culture retains 

the counterstain (the red fuchsin solution) the culture is gram negative. 

F. Describe the quadrant streaking technique to obtain isolated colonies. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 73 of 98

For the most current and official copy, check QMiS. 

Purpose: 

The streak plate technique is the most widely used method of obtaining isolated 

colonies from a mix of cultures. 

Principle: 

The streak plate technique is essentially a method to dilute the number of organisms, 

decreasing the density. This allows for individual colonies to be isolated from other 

colonies. Each colony is considered "pure," since theoretically, the colony began with 

an individual cell. 

Procedure: 

a. Begin with inoculating the first, or primary, quadrant of the agar plate. Use a 

light touch. Don't penetrate or scrape the agar surface. Cover plate with lid. 

Flame the loop, cool by touching an uninoculated portion of the surface. 

b. Now rotate the plate. Open lid and streak again. Remember, the analyst is 

picking up growth from quadrant one, and using this as an inoculum for 

quadrant two. 

c. Flame loop, rotate plate, and repeat procedure for quadrants three and four. 

G. What is the IMViC pattern for E. coli? 

IMViC patterns ++-- (biotype 1) or -+-- (biotype 2) are considered to be E. coli. 

H. What does a typical E. coli isolate look like on L-EMB? 

Dark centered and flat, with or without metallic sheen 

I. When does one use LST-MUG? How does LST-MUG work? 

Use LST-MUG when detecting E. coli in chilled or frozen foods exclusive of bivalve 

molluscan shellfish. 

The LST-MUG assay can presumptively identify E. coli within 24 h. The assay is based 

on the presence of GUD in E. coli, which cleaves the MUG substrate to release 

4methylumbelliferone (MU). When exposed to longwave (365 nm) UV light, MU 

exhibits a bluish fluorescence which is easily visualized. When MUG is incorporated 

into LST medium, coliforms can be enumerated based on gas production from lactose. 

E. coli are presumptively identified by fluorescence in the medium under longwave UV 

light 

J. What time period is allowable between sample shaking and inoculation of tubes? 

Not >15 min should elapse from the time test sample is blended until all dilutions are 

made in prescribed culture media. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 74 of 98

For the most current and official copy, check QMiS. 

Enterohemorrhagic E. coli (EHEC) 

A. Are all STEC also Enterohemorrhagic E. coli (EHEC)? 

No, only a subset of Shiga-toxin producing E. coli (STEC) are involved in causing 

illness in humans and are referred to as enterohemorrhagic E. coli (EHEC). The 

more severe cases are characterized by bloody diarrhea and can progress to 

hemolytic uremic syndrome (HUS) or death. 

B. Do all E. coli strains ferment sorbitol? 

More than 85% of E. coli strains do ferment sorbitol. Significantly, however, almost 

all E. coli O157:H7 do not ferment sorbitol. 

C. Why is tellurite cefixime sorbitol MacConkey agar (TCSMAC) particularly useful for 

the isolation of E. coli O157:H7? 

The incorporation of sorbitol as the primary sugar source in MacConkey agar aids in 

the differentiation of sorbitol negative E. coli O157:H7 (tan to light gray colony 

appearance) from most other E. coli (magenta colony appearance) that do ferment 

sorbitol. 

D. Are all STEC hemolytic? 

Approximately 89 % of STEC and only 15% of non-STEC produce an active 

enterohemolysin and are hemolytic. Therefore the use of agar such as SHIBAM can 

be useful to detect those STEC that are hemolytic. 

E. Why is it important to use multiple differential and selective agars to recover E. coli

O157:H7 and other non-O157 STEC? 

As a group STEC are very phenotypically diverse. The use of several agars that can 

distinguish between these different phenotypic characteristics are useful to improve 

the isolation and recovery of STEC. 

F. Explain why the PCR to confirm the presence of shiga toxin genes (stx) must be done 

as part of the isolate confirmation process even if the PCR was used during the initial 

screening of the food enrichment. 

When the food enrichment is screened for stx genes this is only an indication that 

these gene targets may be present in one or more different genomes and that the 

analysis should continue to isolate and recover one or more STEC strains. After 

recovery it is essential to reconfirm the stx geneotype for each STEC isolate 

recovered individually. 

G. Why is it important to have confirmed STEC isolates further characterized for 

complete virulence profile and serotype by methods such as microarray or whole 

genome sequencing (WGS)? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 75 of 98

For the most current and official copy, check QMiS. 

The assessment of STEC health risk is complicated by the existence of many nonO157 STEC serotypes that do not have the right combination of virulence 

determinants and therefore, do not have the potential to cause severe illness. As a 

result, it is necessary to evaluate the health risk of a particular STEC isolate to human 

health, regardless of serotype, by compiling complete laboratory generated data 

including genetic data that can be evaluated for virulence profiles, serotype and 

similarity to strains involved in human disease. 

Staphylococcus aureus 

A. Describe the symptoms and onset time of staphylococcal food borne disease. 

Primary symptoms may include nausea, vomiting, abdominal cramps, diarrhea, 

headache, and muscular cramps. Secondary symptoms may include retching, 

dehydration, sweating, weakness, and prostration. Symptoms typically develop 

within 6 h after ingestion of contaminated food. Symptoms usually last for 1 to 2 

days. 

B. Describe the difference between intoxication and infection. Which one is associated 

with S. aureus? 

Intoxication is associated with ingestion of pre-formed toxin and does not need 

growth of the organism in the host to produce illness. Infection requires the 

establishment of a pathogen within or on a host after invasion, where the organism 

may begin to grow and cause illness. Staphylococcal foodborne illness is 

considered an intoxication since it does not need growth of the pathogen in the host 

to cause illness. 

C. Which ingredient/s of Baird Parker medium help injured organisms grow? 

This medium contains lithium chloride and tellurite to inhibit the growth of 

accompanying microbial flora, whereas pyruvate and glycine selectively stimulate 

the growth of Staphylococci. 

D. What are typical observations of coagulase positive Staphylococcus aureus when 

plated on Baird Parker medium? What is indicated by the presence or absence of 

a halo around an isolated colony on Baird Parker medium? 

Coagulase positive S. aureus colonies are typically circular, smooth, convex, 2-3 

mm gray to jet-black colonies with an outer clear halo zone surrounding the colony. 

Colonies have a buttery to gummy consistency when touched with a needle. 

Staphylococcus colonies show two characteristic features when grown in Baird 

Parker medium. The first would be characteristic halos which are formed because 

of lipolysis and proteolysis. Second, reduction of tellurite to tellurium producing a 

gray to jet-black coloration. The egg-yolk reaction and tellurite reduction are usually 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 76 of 98

For the most current and official copy, check QMiS. 

found to occur together with a positive coagulase reaction and can thus serve as an 

index for the latter. 

E. Name the staphylococcal enterotoxin type and which is the most common cause of 

food borne disease. 

There are many different designations for SE types on classification schemes 

based on antigenicity. SEA remains the most common toxin implicated in 

foodborne intoxication. 

Coliforms 

A. Why is sodium thiosulfate added to jars used to collect water samples? 

(Investigations Operations Manual (IOM), current edition) To prevent chlorine from 

acting on the bacteria and assures when sample is analyzed, the bacterial load is the 

same as when collected. 

B. Explain the difference between coliforms and fecal coliforms. 

E. coli and the coliforms are both Gram-negative, rod-shaped facultatively anaerobic 

bacteria, however, coliforms produce gas from glucose (and other sugars) and 

ferment lactose to acid and gas within 48 h at 35°C, but fecal coliforms (and E. coli as 

a coliform) produce gas from glucose (and other sugars) and ferment lactose to acid 

and gas within 48 h at 45.5°C. Also, coliforms are found throughout the environment 

and fecal coliforms are found in the intestinal tract of warm-blooded vertebrae. 

C. What organisms are considered coliforms? 

Escherichia, Enterobacter, Klebsiella, Serratia, Citrobacter, Proteus. 

Aerobic Plate Count 

A. Name foods that may have natural high counts. Name foods that should have low 

counts. 

One example of a high natural APC count would be sprouts. An example of a food with 

a low natural APC count would be tree fruits such as oranges and lemons. 

B. Why are APC counts of interest to FDA? 

The aerobic plate count (APC) is intended to indicate the level of live microorganisms 

in a product at the time of analysis. By modifying the temperature of incubation, the 

analyst can selectively screen for microorganisms such as 

thermophiles/mesophiles/psychrotrophiles, to name a few. 

C. Why do "spreaders" sometimes form when doing an APC? At the air/agar interface? At 

the agar/glass interface? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 77 of 98

For the most current and official copy, check QMiS. 

Spreading colonies are usually of 3 distinct types: 1) a chain of colonies, not too 

distinctly separated, that appears to be caused by disintegration of a bacterial clump; 

2) one that develops in film of water between agar and bottom of dish; and 3) one that 

forms in film of water at edge or on surface of agar. If plates prepared from sample 

have excessive spreader growth so that (a) area covered by spreaders, including total 

area of repressed growth, exceeds 50% of plate area, or (b) area of repressed growth 

exceeds 25% of plate area, report plates as spreaders. 

D. How long and at what temperature can one "thaw" a frozen food? Discuss. 

Normally, a sample can be thawed at 2-5°C within 18 h. If rapid thawing is desired, 

thaw the sample at less than 45°C for not more than 15 min. When thawing a sample 

at elevated temperatures, agitate the sample continuously in thermostatically 

controlled water bath. 

E. Will there by a change to the APC results with repeated freezing and thawing of product? 

One theory is that the viability and subsequent growth on agar plates of the organisms 

may be compromised due to disruption and destruction of bacterial DNA through 

repeated freezing and thawing. 

F. What is the proper procedure for manually mixing dilution blanks? 

Shake all dilutions 25 times in 30 cm (1 ft) arc within 7 seconds. 

G. At what temperature should pour plates be dispensed? 

Desired temperature is 45 ± 1°C. 

H. How should the analyst interpret plates with more than 250 colony-forming units 

(CFU’s)? 

When number of CFU per plate exceeds 250, for all dilutions, record the counts as too 

numerous to count (TNTC) for all but the plate closest to 250, and count CFU in those 

portions of plate that are representative of colony distribution. See BAM Chapter 3 for 

detailed guidelines. Mark calculated APC with EAPC to denote that it was estimated 

from counts outside 25-250 per plate range. When plates from both dilutions yield 

more than 250 CFU each (but fewer than 100/cm2), estimate the aerobic counts from 

the plates (EAPC) nearest 250 and multiply by the dilution. 

Example: 

Colonies 

1:100 1:1000 EAPC/ml (g) 

TNTC 640 640,000 

TNTC = too numerous to count. EAPC = estimated aerobic plate 

count. 

I. How should the analyst interpret plates with less than 25 CFU’s? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 78 of 98

For the most current and official copy, check QMiS. 

When plates from both dilutions yield fewer than 25 CFU each, record actual plate 

count but record the count as less than 25 x 1/d when d is the dilution factor for the 

dilution from which the first counts were obtained. 

Example: 

Colonies 

1:100 1:1000 EAPC/ml (g) 

18 2 <2,500 

0 0 <2,500 

Yeast and Mold Count 

A. Why are yeast and mold count of interest to FDA? 

Both yeasts and molds can cause various degrees of deterioration and 

decomposition of foods. Also, several foodborne molds and some yeasts can produce 

mycotoxins which are hazardous to human and animal health. 

B. What is the medium of choice for yeast and mold analysis? 

Potato Dextrose Agar. 

C. What medium is especially useful for analyzing samples containing "spreader" 

molds? 

Potato Dextrose-Salt Agar. 

D. What agent/agents are added to the agar to inhibit bacterial growth? 

Antibiotics (Chlortetracycline-HCl, Chloramphenicol, or Streptomycin) or Sterile 10% 

Tartaric Acid. 

E. Why should the plates be left undisturbed until the incubation period is complete? 

Handling plates could result in secondary growth from dislodged spores which would 

make counts invalid. 

INTERMEDIATE (TIER 2) ORGANISMS 

Vibrio spp. 

A. Describe the foodborne illness and onset times of the Vibrio spp. that were studied. 

 Vibrio cholerae: 

Cholera is an acute, diarrheal illness caused by infection of the intestine with the 

bacterium Vibrio cholerae. The infection is often mild or without symptoms, but 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 79 of 98

For the most current and official copy, check QMiS. 

sometimes it can be severe. Symptoms of Asiatic cholera may vary from a mild, 

watery diarrhea to an acute diarrhea, with characteristic rice water stools. 

Additional symptoms may include vomiting, leg cramps, nausea, dehydration, and 

shock. After severe fluid and electrolyte loss, death may occur. Onset of the illness 

is generally sudden, with incubation periods varying from 6 hours to 5 days. 

Symptoms usually last 2 to 3 days although in some patients they can continue up 

to 5 days. Most people are infectious from the onset of illness until a few days after 

recovery. 

Vibrio parahaemolyticus: 

When ingested, V. parahaemolyticus causes watery diarrhea often with abdominal 

cramping, nausea, vomiting fever and chills. Usually these symptoms occur within 

24 hours of ingestion. Illness is usually self-limited and lasts 3 days. Severe 

disease is rare and occurs more commonly in persons with weakened immune 

systems. V. parahaemolyticus can also cause an infection of the skin when an 

open wound is exposed to warm seawater. 

Vibrio vulnificus: 

V. vulnificus can cause disease in those who eat contaminated seafood or have an 

open wound that is exposed to seawater. Wound infections may lead to skin 

breakdown and ulceration. Among healthy people, ingestion of V. vulnificus can 

cause vomiting, diarrhea, and abdominal pain. (In healthy individuals, 

gastroenteritis usually occurs within 16 hours of ingesting the organism). In 

immunocompromised persons, particularly those with chronic liver disease, V. 

vulnificus can infect the bloodstream, causing a severe and life-threatening illness 

characterized by fever and chills, decreased blood pressure (septic shock), and 

blistering skin lesions. V. vulnificus bloodstream infections are fatal about 50% of 

the time. 

B. What are the advantages and disadvantages of using a PCR method? 

Advantages: PCR analysis is rapid and can be performed within 48 hours including 

sample enrichment. Only enterotoxigenic Vibrio spp. will yield a positive result. It will 

detect enterotoxigenic V. mimicus that may be missed in the standard BAM 

bacteriological analysis. (Colonies of V. mimicus appear green on TCBS selective 

media). 

Disadvantages: A positive PCR result does not indicate the viability of the organisms 

detected. (Therefore, viable cells containing the ctxAB target sequence may not be 

able to produce enterotoxin in culture media. Certain food matrices may inhibit PCR 

reactions and yield false negative results. A positive food spike control should be 

analyzed with each new food tested.) 

C. Why is NaCl added to media used for Vibrio spp.? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 80 of 98

For the most current and official copy, check QMiS. 

Vibrio spp. other than V. cholerae and V. mimicus are halophilic and do not grow in 

media that lack NaCl. 

D. Why do some methods enumerate the organism and other methods only check for 

the presence of the organism? 

There are guidance tolerance levels for some Vibrio spp. organisms and zero 

tolerance for others. For example, enterotoxigenic V. cholerae has a zero-tolerance 

level whereas violative levels of V. parahaemolyticus are ≥ 1 X 104 organisms/gram 

food. 

E. What are the characteristics of V. mimicus? What are the similarities and differences 

of this organism compared to V. cholerae? 

V. mimicus colonies are sucrose negative on TCBS agar whereas V. cholerae are 

sucrose positive. V. mimicus are also urease negative. V. cholerae are urease 

positive. 

F. What are the major factors in the pathogenesis of V. cholerae, V. parahaemolyticus, 

V. vulnificus, and V. mimicus? 

V. cholerae- cholera toxin (CT) or cholera-like toxin (non01/0139). 

V. parahaemolyticus - produces a thermostable direct hemolysin also referred to as 

the Kanagawa phenomenon-associated hemolysin. Epidemiological evidence 

suggests that it is a virulence factor. All strains produce thermolabile hemolysin 

(species specific). Some produce thermostable direct hemolysin (TDH) some produce 

thermostable related hemolysin (TRH). Some produce both. V. vulnificus - produces 

cytotoxin/hemolysin. 

V. mimicus - produces a cholera-like toxin. 

Discussion: Cholera symptoms result from the action of cholera toxin (CT), which is a 

chromosomally mediated, heat-labile enterotoxin. CT has a high level of similarity to 

the plasmid mediated, heat-labile enterotoxin (LT) of Escherichia coli. CT is an 

oligomeric protein composed of an A subunit and five B subunits. The B subunits bind 

to ganglioside GM1 receptors on the membranes of intestinal epithelial cells, while 

the A subunit activates adenylate cyclase, producing increased levels of cyclic AMP 

and resulting in the hypersecretion of water and electrolytes. The outpouring of water 

and electrolytes into the lumen of the intestine causes copious purging, resulting in 

the severe dehydration associated with the classical clinical presentation of cholera. 

With rare exceptions, Non-01 and 0139 V. cholerae strains do not produce CT and 

rarely cause epidemic disease. Several other toxins have been identified from non01/0139 strains. These include a hemolysin stable enterotoxin very similar to that 

produced by E. coli referred to as NAG-ST; and a hemolysin termed NAG-rTDH 

which is related to the thermostable direct hemolysin (TDH) of V. parahaemolyticus. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 81 of 98

For the most current and official copy, check QMiS. 

Bacillus cereus 

A. Describe the symptoms and onset times for the two types of B. cereus food borne 

diseases. What foods have been implicated in each type? 

Type 1: Diarrheal type – diarrhea, abdominal pain, nausea, some vomiting, usually 

fever with an onset time usually 816 hours. Implicated foods include but are not 

limited to meats, vegetables, milk, cream pastries, and soups. Type 2: Emetic type – 

primary symptom is vomiting with and onset time usually between 1 – 5 hours. 

Implicated foods include but are not limited to fried or cooked rice and another 

starchy food such as potatoes. 

B. Can B. thuringiensis produce diarrheal antigens? Diarrheal disease? 

Yes. 

C. The symptomatic profile of emetic type of food poisoning produced by some strains of 

B. cereus most closely mimics that of Clostridium pefringens or Staphylococcus 

aureus? 

Staphylococcus aureus intoxication. 

D. The toxins of B. cereus most commonly associated with food poisoning are: 

Diarrheal and emetic components. 

E. Explain the purpose for each ingredient used in MYP agar and how B. cereus can be 

interpreted on MYP agar. 

Ingredients included in MYP agar include beef extract, mannitol, peptone, NaCl, 

phenol red, and agar. Check plates for lecithinase production as indicated by zone of 

precipitation surrounding growth. Mannitol is not fermented by isolate if growth and 

surrounding medium are eosin pink. (Yellow color indicates that acid is produced from 

mannitol.) B. cereus colonies are usually lecithinase-positive and mannitol-negative 

on MYP agar. 

F. When is the Plate Count Method recommended? When is Most Probable Number 

(MPN) recommended? 

The MPN technique is recommended for enumerating B. cereus in foods that are 

expected to contain fewer than 10 B. cereus organisms/g. It may also be preferred for 

examining certain dehydrated starchy foods for which the plate count technique is not 

recommended. 

G. When interpreting test results (motility, hemolytic activity, plating characteristics, and 

crystal production) what are typical results for each pertaining to B. cereus? 

Most strains of B. cereus are motile; however, some non-motile strains exist. B. 

cereus exhibit hemolysis (sheep RBC). B. cereus exhibit development of rhizoid 

growth, which is characterized by production of colonies with long hair or root-like 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 82 of 98

For the most current and official copy, check QMiS. 

structures that may extend several centimeters from site of inoculation. Rough 

galaxy-shaped colonies are often produced by B. cereus strains and should not be 

confused with typical rhizoid growth, which is the definitive characteristic of B. 

mycoides. B. cereus and other members of the B. cereus group do not produce 

protein toxin crystals. B. thuringiensis usually produces protein toxin crystals that can 

be detected by the staining technique either as free crystals or parasporal inclusion 

bodies within the exosporium. 

Campylobacter 

A. Describe the symptoms and onset time of Campylobacter food borne disease. 

Onset of disease is 2-10 days. Symptoms include fever of greater than 40°C, 

abdominal pain, nausea, vomiting, and mild to severe diarrhea. Diarrhea is selflimiting, 2-7 days. 

B. Describe methods used to obtain microaerobic conditions. 

Microaerobic conditions can be achieved by bubbling with or shaking in a modified gas 

mixture of 5% O2, 10% CO2, 85% N2. Anaerobe jars evacuated with the modified gas 

mixture or containing a Campy Pak or large jars with a single Anaerobe Pak will also 

provide a microaerobic environment. 

C. What are the oxygen-quenching compounds added to Campylobacter media? 

FBP (ferrous sulfate, sodium metabisulfite, sodium pyruvate), hemin, blood, and 

charcoal are oxygen-quenching compounds. 

D. Which biochemical test is used to differentiate between C. jejuni and C. coli? 

Hippurate hydrolysis. 

E. What are the advantages and disadvantages of using rapid methods such as the Dryspot 

Campy Test or Alert for Campylobacter? 

The Dryspot Test gives a quick confirmation that the analyst has isolated one of the 

most commonly found Campylobacters (C. jejuni, C. coli, C. lari, C. upsaliensis, C. 

fetus); however, it will not serotype the isolate nor speciate it. Additionally, it will not 

react with all C. fetus strains. (FYI-The reference to Alert will be removed from future 

BAM/LPM editions.) 

Yersinia enterocolitica

A. Describe the symptoms and onset time of Yersinia enterocolitica illness. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 83 of 98

For the most current and official copy, check QMiS. 

Symptoms of yersiniosis include gastroenteritis with diarrhea and/or vomiting. The 

usual onset time is 4 to 7 days after exposure with the illness lasting 1 to 3 weeks or 

longer. Right-sided abdominal pain and fever are the hallmark symptoms. 

B. Does Yersinia grow and survive during refrigerated storage? 

Yersinia may survive and grow during refrigerated storage. 

C. What enrichment broths and selective media are used to culture Yersinia enterocolitica? 

Yersinia enterocolitica is enriched in peptone sorbitol bile broth (PSBB) or on spread 

plate (MacConkey and CIN - Cefsulodin Irgasan Novobiocin agars) if high levels are 

suspected. 

D. What enrichment broths and selective media are used to culture Yersinia

pseudotuberculosis? 

Y. pseudotuberculosis is enriched in PMP broth and streaked onto MacConkey and 

YSA/CIN agar plates. 

E. Describe the biochemical characteristics of Yersinia enterocolitica and Yersinia

pseudotuberculosis. 

Y. enterocolitica: Characteristic biochemical reactions include anaerogenic 

fermentation of glucose, positive urease, and positive motility below 30°C but negative 

at 35°C. Y. enterocolitica are variable in their indole reaction; they do not ferment 

lactose, and do not utilize citrate. They are also phenylalanine deaminase negative 

and lysine decarboxylase negative. 

Y. pseudotuberculosis: Characteristic biochemical reactions include fermentation of 

glucose, positive urease, and positive motility below 30°C but negative at 35°C. Y. 

pseudotuberculosis is indole negative; they do not ferment ornithine, sorbitol and 

sucrose, and do not utilize citrate. They are also phenylalanine deaminase negative 

and lysine decarboxylase negative. They can ferment mannitol and rhamnose. 

F. What is the relationship of plasmids and Yersinia? 

Yersinia spp. that cause human yersiniosis carry a plasmid (41- 50Mdal). 

G. What types of tests are used to determine pathogenicity? 

There are several traits related to virulence: 

 autoagglutination in certain media at 35°C-37°C 

 inhibition of growth in calcium deficient media 

 binding of crystal violet dye at 35°C-37°C 

 increased resistance to normal human sera 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 84 of 98

For the most current and official copy, check QMiS. 

 production of a series of outer membrane proteins at 35°C-37°C ability to 

produce conjunctivitis in guinea pig or mouse (Serenytest) 

 lethality in adult and suckling mouse by intraperitoneal injection of live organisms 

Clostridium perfringens 

A. Describe the symptoms and onset time for C. perfringens foodborne disease. 

Affected individuals usually only develop diarrhea and severe abdominal pain. Less 

common side effects are vomiting, fever, shivering and headache. Onset usually will 

develop between 8 to 14 hours after ingestion of contaminated food and usually 

resolve spontaneously within 12 to 14 hours. 

B. How does one test the organism for toxin production? 

Utilize a specially designed enterotoxin toxin testing kit such as Reverse Passive 

Latex Agglutination (RPLA) kit. 

C. Tell when each enumeration method would be use. 

Laboratory capability will determine enumeration method. Analysts are to always 

utilize official validated methodology 

D. How do anaerobe jar commercial systems produce anaerobic conditions? How can 

one tell if it worked? 

Inoculated media is placed in the anaerobe jar with or without palladium catalyst and 

with a generator envelope. The system is then sealed with an airtight lid. The 

envelopes are H2 + CO2 generator envelopes with or without a palladium catalyst. 

Note* catalyst is to be in envelope or jar for successful conversion to anaerobic 

conditions. Some systems either have the catalyst in the lid of the jar, or the catalyst 

is contained in the generator envelope. To visually see if anaerobic conditions are 

produced through the anaerobic container system, there are commercially 

purchasable indicator strips which will turn colors (usually blue when exposed to 

oxygen and will turn white when O2 is depleted from the system. 

E. Describe stormy fermentation and how to test C. perfringens for stormy fermentation? 

After inoculation into media such as Iron Milk Medium (M68), coagulation of milk to 

produce an acid curd with subsequent disruption or fracturing of the spongy curd by a 

large volume of gas produced by the organism exhibits stormy fermentation. 

F. Describe key characteristics of C. perfringens including motility, gram-reaction, nitrate 

reduction, and lecithinase activity. 

Non-motile. 

Gram-positive spore-forming Bacillus 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 85 of 98

For the most current and official copy, check QMiS. 

Reduce nitrates to nitrites 

C. perfringens colonies on TSC/EY - 2-4 mm opaque, white zone surrounding 

Exhibits lecithinase activity 

Clostridium botulinum 

A. What are the symptoms and onset time of botulism? 

The classic symptoms of botulism include double vision, blurred vision, drooping 

eyelids, slurred speech, difficulty swallowing, dry mouth, and muscle weakness. 

Infants with botulism appear lethargic, feed poorly, are constipated, and have a weak 

cry and poor muscle tone. These are all symptoms of the muscle paralysis caused by 

the bacterial toxin. If untreated, these symptoms may progress to cause paralysis of 

the arms, legs, trunk and respiratory muscles. In foodborne botulism, symptoms 

generally begin 18 to 36 hours after eating a contaminated food, but they can occur 

as early as 6 hours or as late as 10 days. 

B. What is wound botulism and infant botulism? 

Wound botulism is caused by toxin produced from a wound infected with Clostridium 

botulinum. Infant botulism is caused by consuming the spores of the botulinum 

bacteria, which then grow in the intestines and release toxin. 

C. Name the different types of toxins and which have been implicated in human 

botulism. 

There are seven types of botulism toxin designated by the letters A through G; only 

types A, B, E and F cause illness in humans. 

D. Describe how the MLD is calculated. 

Dilutions that kill and dilutions that do not kill are needed to establish an endpoint or 

the minimum lethal dose (MLD) as an estimate of the amount of toxin present. The 

MLD is contained in the highest dilution killing both mice (or all mice inoculated). 

From these data, the number of MLD/ml can be calculated. 

E. What is the difference between preformed and formed toxin? 

Preformed toxin is when botulin toxin is detected in a suspect food, whereas formed 

toxin is when botulin toxin is produced during growth in a culture medium. 

Alkaline Phosphatase 

A. Why is the presence of alkaline phosphatase determined in dairy products? 

To detect improper pasteurization, a negative phosphatase test indicates most of the 

milk used has been properly heated during pasteurization. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 86 of 98

For the most current and official copy, check QMiS. 

B. What is the reason for heating the control blank? 

The control blank is heated to identify the amount of color development which may be 

due to reagent contamination and/or interfering color material in the sample so that it 

can be subtracting from sample colorimetric readings ensuring the remaining color 

development can be attributed to the presence of alkaline phosphatase. 

C. Why is a buffer used in the analysis? 

To maintain optimal pH of between 9.85 and 10.2 for the reaction to occur. 

D. In the AOAC confirmation method, why are different buffer and precipitant 

concentrations used for different cheeses? 

Different strength buffers are used for different types and ages of cheese. 

E. What parameters need to be controlled during analysis for the phenol to be liberated 

from the disodium phenyl phosphate substrate? 

A pH range of 9.85 to 10.2 and incubation time. 

F. To what compound is the alkaline phosphatase enzyme activity proportional? 

Phenol. 

G. Describe the reaction that takes place for phenol to be measured colorimetrically? 

To measure phenol either 2,6-dichloroquinonechlorimide (CQC) in the screening 

method or 2,6 dibromoquinonechlorimide (BQC) in the confirmation method reacts in 

the presence of catalyst (CuSO4) to form indophenol blue. The indophenol blue can 

then be measured colorimetrically. 

Canned Food and Can Seam Examination 

A. What is a Flipper, Springer, Soft Swell and Hard Swell? 

Flipper: A can that normally appears flat. When brought down sharply on its end on a 

flat surface, one end flips out. When pressure is applied to this end, it flips in again and 

the can appears flat. Springer: A can with one end permanently bulged. When 

sufficient pressure is applied to this end, it will flip in, but the other end will flip out. Soft 

Swell: A can bulged at both ends, but not so tightly that the ends cannot be pushed in 

somewhat with thumb pressure. Hard Swell: A can bulged at both ends, and so tightly 

that no indentation can be made with thumb pressure. The next stage is the bursting of 

the can. 

B. What is flat-sour spoilage? What bacteria can cause this? 

A type of microbial spoilage characterized by a markedly lowered pH, sour and 

abnormal odor, and sometimes cloudy liquor. Gas is not usually produced therefore 

the can remains flat, hence the name “flat-sour”. Flatsour organisms include Bacillus
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 87 of 98

For the most current and official copy, check QMiS. 

thermacidurans (Bacillus coagulans) and Bacillus stearothermophilis, both being 

thermophilic bacteria, growing and causing flat-sour spoilage at elevated temperatures 

(50-55oC). 

C. Does microbial spoilage of a food always result in a swollen can? Explain microbial 

spoilage of a food does not always result in a swollen can. 

The following are examples. 

Flat-sour spoilage as explained in question #2. 

Sulfide spoilage: H2S gas is produced which is absorbed (soluble) by the product. 

Usually blackened, “rotten egg” odor. The can remains flat. Thermophilic, spore 

forming anaerobes are primarily responsible for this spoilage. Clostridium nigrificans is 

considered the type species of this group. 

Proteolytic bacteria such as Clostridium botulinum Type A may not produce enough 

gas to swell the can. 

D. Why are certain canned food media incubated at 55oC? 

Media is incubated at 55oC to detect thermophilic bacteria which cause flat-sour and 

sulfide spoilage. 

E. Explain the microbiology and physical attributes of each of the following: 

Underprocessing, leaker spoilage, hydrogen swell and overfill. Underprocessing 

caused by undercooking. Either the temperature and/or time of the cook were short. 

This is the most serious processing problem. Characterized by pure cultures of Gram + 

rods and intact seams. If cans missed the retort altogether, there could be a high rate 

of swell and a mixed flora of rods and cocci. Leaker Spoilage: Caused by postprocessing contamination, usually characterized by defective seams, microleaks and a 

mixed flora of rods and cocci. Hydrogen Swell: Caused by the acid of the product 

reaction with the metal of the can, resulting in hydrogen gas. Most prevalent in acid 

foods such as canned tomatoes which have exceeded the normal shelf life of the 

product. Overfill: Caused by too much product filled into the can, resulting in not 

enough headspace and lack of proper vacuum. 

F. Explain the construction of a metal can. Name the components of the double seam. 

The metal can is made of either steel or aluminum. It consists of the can body and two 

lids (3 piece can), or the can body and one lid (2 piece drawn can). The can body is 

joined together by a weld. The interior of a steel can is usually coated with a protective 

enamel, which inhibits rusting and interaction of the product with the steel. The lid is 

attached by a double seaming operation. The double seam consists of 5 thicknesses 

of plate plus a thin layer of sealing compound. The seam is formed by two rolling 

operations. 

G. Explain water activity and its significance in an acidified and low acid canned food. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 88 of 98

For the most current and official copy, check QMiS. 

Water activity is a number which provides information about the vapor pressure of 

water in a system. It’s measured by dividing the equilibrium vapor pressure of the 

product by that of pure water, which is taken to be 1.0. The higher the water activity 

(Aw), the more free water there is for microbial growth. Water activity is part of the 

definition of a Low Acid Canned Food and Acidified Low Acid Canned Food. In both, 

according to 21 CFR, parts 113 and 114. the Aw must be >0. 

Cronobacter spp. 

A. What species belong to genus Cronobacter and which species have been involved in 

human illnesses? 

Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter 

muytjensii, Cronobacter dublinensis, and Cronobacter genomospecies I (later renamed 

as Cronobacter universalis). All species, except Cronobacter genomospecies I, have 

been involved in clinical cases. 

B. What is the morphology of typical colonies on chromogenic agars? 

Presumptive Cronobacter colonies on DFI agar appear either dark green, weak green, 

or brownish green. Some colonies only have a green center with a white/yellow border. 

Presumptive Cronobacter colonies on R&F agar appear blue to black, or blue to grey 

with the red background. The red background can appear purplish red with different 

strain or under different light conditions. Cronobacter does not change the color of R&F 

agar, but in the presence of background microflora which change the color of R&F 

agar from red to yellow, Cronobacter colonies appear blue to green. 

C. Can the method be performed without running the qPCR assay? 

The cultural portion of this method is a complete detection/isolation method, so it can 

be used as a stand-alone method if PCR technology is unavailable. The PCR portion 

of the method is a screening method, whose positive results should always be 

confirmed with the cultural method. The PCR method may be used to confirm pure 

cultures as Cronobacter spp. 

Cosmetic Analysis 

A. Describe the sample preparation techniques for liquids and semi powders; solids and 

powders; preparations with petroleum base; aerosols of powders and liquids; and 

aerosols of soaps and other foamy liquids. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 89 of 98

For the most current and official copy, check QMiS. 

Liquids: Decimally dilute 1 ml liquid directly into 9 ml modified letheen broth (MLB) in 

20 x 150 mm screw-cap test tube for the 10-1 dilution. Solids and powders: 

Aseptically remove and weigh 1 g sample into 20 x 150 mm screw-cap test tube 

containing 1 ml sterile Tween 80. Disperse product in Tween 80 with sterile spatula. 

Add 8 ml sterile MLB and mix thoroughly. This will be the 10-1 dilution. Cream and 

oil-based products: Aseptically remove and weigh 1 g sample into 20 x 150 mm 

screw-cap tube containing 1 ml sterile Tween 80 plus five to seven 5-mm glass beads 

(or ten to fifteen 3-mm glass beads). Mix total contents with Vortex mixer. Adjust total 

volume to 10 ml with sterile MLB (8 ml) for the 10-1 dilution. Aerosols of powders, 

soaps, liquids, and other materials: Decontaminate nozzle of spray can as much as 

possible by swabbing with gauze pad moistened with 70% (v/v) aqueous ethanol. 

Expel some product to flush out nozzle; then spray reweigh. This will be a 10-1 

dilution if exactly 1 g of sample was obtained. 

B. What is the purpose of dilutions and other added ingredients? 

Dilutions and other added ingredients partially inactivate preservative systems 

commonly found in cosmetic products. 

C. How do Pseudomonas and Klebsiella differ biochemically? What characteristics 

clearly separate them? 

Pseudomonas is a non-fermenting microorganism and Klebsiella is a fermenter. 

Pseudomonas can be separated from Klebsiella in that it is oxidase positive and does 

not ferment glucose while Klebsiella is oxidase negative and ferments glucose. 

D. If, biochemically, a culture was indicated to be Pseudomonas but it did not produce a 

fluorescent yellow or blue pigment, would a microbiologist still consider the culture as 

Pseudomonas? Explain. 

The culture could still be a Pseudomonas based on other identifying characteristics. 

Production appears to be somehow involved in iron acquisition. Approximately 90% 

of Pseudomonas spp. are fluorescent. The pigments are useful differentiating 

markers. The classification of Pseudomonas aeruginosa is based on the production 

of a number of pigments including a pyocyanin (blue) and fluorescein (yellowish 

green). Some strains also produce pyorubin (red), pyomelanin (brown), two other red 

pigments (aeruginosin A and B) and various other pigments. 

E. Which organisms are considered pathogenic in the eye area? On the skin? 

Pathogens or opportunistic pathogens whose incidence would be of particular 

concern, especially in eye-area cosmetic products, include S. aureus, Streptococcus 

pyogenes, P. aeruginosa and other species, and Klebsiella pneumoniae. Some 

microbes normally regarded as nonpathogenic may be opportunistically pathogenic 

(e.g. in wounds). 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 90 of 98

For the most current and official copy, check QMiS. 

Nutritional and Dietary Supplements 

A. What are the controls used in this experiment? 

Controls used: for Total Aerobic Microbial Count, inoculate duplicate plates with 25–

250 cfu of Staphylococcus aureus (ATCC No. 6538), Escherichia coli (ATCC No. 

8739), and Bacillus subtilis (ATCC No. 6633) to demonstrate a greater than 70% 

bioburden recovery in comparison to a control medium. For the Sabouraud Dextrose 

Agar Medium used for Total Combined Molds and Yeasts Count, inoculate duplicate 

plates with 25–250 cfu of Candida albicans (ATCC No. 10231) and Aspergillus 

brasiliensis (ATCC No. 16404) to demonstrate a greater than 70% bioburden recovery 

in comparison to a control medium. For Enterobacterial Count (Bile-Tolerant GramNegative Bacteria), appropriate dilutions of Escherichia coli (ATCC No. 8739) and 

Salmonella typhimurium (ATCC No. 13311) are used. 

B. What are some ways to neutralize inhibitory substances? 

The following are examples of ingredients and their concentrations that may be added 

to the culture medium to neutralize inhibitory substances present in the sample: soy 

lecithin, 0.5%; and polysorbate 20, 4.0%. Alternatively, repeat the test as described in 

the preceding paragraph, using Fluid Casein Digest–Soy Lecithin–Polysorbate 20 

Medium to demonstrate neutralization of preservatives or other antimicrobial agents in 

the test material. Where inhibitory substances are contained in the product and the 

latter is soluble, a suitable, validated adaptation of a procedure set forth under 

Procedure using the Membrane Filtration Method may be used. 

If, despite the incorporation of suitable inactivating agents and a substantial increase in 

the volume of diluent, it is still not possible to recover the viable cultures described 

above, and where the article is not suitable for the employment of membrane filtration, 

it can be assumed that the failure to isolate the inoculated organism is attributable to 

the bactericidal or bacteriostatic activity of such magnitude that treatments are not able 

to remove the activity. This information serves to indicate that the article is not likely to 

allow proliferation or contamination with the given species of microorganism. 

Monitoring should be continued in order to determine the inhibitory range and 

bactericidal activity of the article. 

C. What is Purified Water? 

Purified Water is water obtained by distillation, ion-exchange treatment, reverse 

osmosis, or other purifying process. It is prepared from water complying with the 

regulations of the federal Environmental Protection Agency with respect to drinking 

water. It contains no added substance. 

D. How does one determine what microbial test to run on products? 

The microbial test run is that specified by the USP monograph for that product. 

E. What is the preparatory test? 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 91 of 98

For the most current and official copy, check QMiS. 

The preparatory test is done to demonstrate that the test specimens do not of 

themselves, inhibit the multiplication, under the test conditions of microorganisms that 

may be present. 

ADVANCED (TIER 3) ORGANISMS 

Viruses 

A. List two ways that viruses differ from bacteria? 

1. Viruses require a host or cell line for replication where bacteria can replicate 

outside the host. 

2. Viruses contain a capsid or protein coat. 

B. What is the genetic material found in viruses? 

Viruses contain either RNA or DNA, their genetic material can either be single 

stranded or double stranded. 

C. What process is required before RNA viruses can be detected by qPCR? 

RNA Viruses must be converted to cDNA (complimentary DNA) during a process 

called reverse transcriptase before the gene can be amplified via PCR. 

D. How are viruses removed and concentrated from fruit, finfish, and molluscan shellfish? 

Fruit and finfish are surface contaminated and are rinsed in a filter bag at a high pH to 

remove the small virus particles from the product. The eluted virus can fit through the 

filter and can be removed physically removed from large food particles. Moderate 

centrifugation separates the even smaller food particles. Molluscan shellfish are filter 

feeders and viruses concentrated in their digestive glands. The digestive glands are 

dissected from the rest of the animal and composited from many animals. This 

composite is then homogenized, and treated through series of absorption, elution, and 

ultra-speed centrifugation to concentrate the virus. A chloroform extraction step is 

used on the concentrated samples to remove organic matter prior to a second series of 

absorption, double elution, and concentration. Internal contamination allows for more 

inhibitors to be mixed with the sample concentrate and this method uses multiple 

cycles of Ultracentrifugation and double elution of the chloroform cleanup to reduce 

these inhibitors. 

E. What is the importance of pH in removing viruses from foods and what characteristic of 

the target virus is important in this approach? 

The isoelectric point of a virus is the pH at which it absorbs to food particles. This pH 

is used to determine the pH needed for various steps of the extraction and 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 92 of 98

For the most current and official copy, check QMiS. 

concentration steps to release the virus from the food (higher pH) or absorb (lower pH) 

the virus to foods. 

F. How do you determine if the amount of inhibition is too high to be able to rely on negative 

PCR results for a given virus target? 

If the average Ct value (cycle # at which target detection fluorescence crosses the predefined threshold set by the method) of the IAC (internal control) in the sample wells is 

more than 4 cycles delayed from the Ct of the internal control in the No Template 

Control (NTC) then there is too much inhibition to rely on a negative result for the 

target organism and the inhibition must be resolved as per the method. 

Cyclospora 

A. Explain why methods based on microscopic examination cannot be used reliably to 

detect and characterize C. cayetanensis in food and agricultural water. 

https://www.fda.gov/food/laboratory-methods-food/bam-chapter-29-cronobacter 

Microscopy lacks the sensitivity and specificity to detect low concentrations of oocysts 

or differentiate between morphologically identical species of Cyclospora. 

B. Explain why person to person transmission of C. cayetanensis does not occur. 

Individuals infected with C. cayetanensis shed unsporulated oocysts in their stools. 

These unsporulated oocysts are not infective when freshly passed and require a week 

or longer in the environment at optimal temperatures to sporulate and become 

infective. 

C. Explain the main steps included in the process for molecular identification of C. 

cayetanensis in fresh produce by PCR as described in BAM Chapter 19b. 

The process for molecular identification of C. cayetanensis in fresh produce by PCR 

can be broken down into the following steps: 1. Produce wash procedure, 2. Isolation 

of DNA from produce wash, and 3. Identification by PCR amplification. 

D. Explain the main steps included in the process for molecular identification of C. 

cayetanensis in agricultural water by PCR as described in BAM Chapter 19c. 

1. Collect 10L sub-samples using the dead-end ultrafiltration procedure described in 

part I of BAM 19c (Dead-end Ultrafiltration). 

2. C. cayetanensis oocysts are recovered from ultrafilters by backflushing and 

concentration into a pellet following the protocol of Part II of 19c. 

3. DNA from recovered oocysts within the pellet is extracted using the FastPrep-24 

bead beater and the FastDNA SPIN Kit for Soil. 

4. Detection of C. cayetanensis DNA is performed using a species-specific qPCR assay 

targeting the 18S rRNA gene. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 93 of 98

For the most current and official copy, check QMiS. 

Whole Genome Sequencing (WGS) 

A. Describe general steps for WGS analysis of bacterial pathogens. 

Extract and quantitate DNA from a single colony from a pure culture bacterial isolate. 

Dilute input DNA and tagment DNA fragments. Generally, this process uses a 

transposase enzyme to simultaneously fragment and inserts adapter sequences into 

dsDNA. Attach specified index primers to fragments by PCR amplification. PCR 

cleanup to remove unincorporated index primers and purify amplified product. Library 

normalization. GenomeTrakr protocol uses a bead-based approach and PulseNet 

protocols use a quantitation and dilution approach. This step is intended to refine the 

library size and prepare appropriate concentrations from each isolate to be included in 

the sequencing pool. Pool libraries from all isolates to be included in sequencing run 

and load onto sequencing cartridge. Load cartridge and flow cell on MiSeq instrument 

and initiate sequencing run. After sequencing run has completed check basic run 

quality metrics. This includes cluster density, Q30 and approximate genome coverage 

for each isolate included in the run. Transfer data for upload to NCBI. Wash and 

maintain MiSeq instrument between sequencing runs. 

B. What is the main difference between DNA extraction procedures for Gram positive and 

Gram-negative isolates? 

The extraction of Gram-positive organisms includes an additional enzymatic lysis step 

using lysozyme. 

C. Why should index primers be rotated between sequencing runs? 

Index primer combinations should be rotated to avoid sample carryover and crosscontamination from a previous run. 

D. What is the basic sequence coverage requirement for Salmonella, Listeria and E. coli? 

Salmonella - 30X, Listeria – 20X, and E. coli – 40X 

E. How often should you wash your sequencer? What should you wash your machine 

with? 

Each lab has their own local variations to this process so please check local guidance 

or with your local SME. Illumina provides minimal requirements as follows: the Miseq 

will prompt you to perform a post-run wash after each sequencing run. Additionally, 

they recommended maintenance washes to be performed at least monthly, a standby 

wash to be performed if the instrument will remain idle for a least 7 days and every 30 

days after while it remains idle. Many labs have elected to run a maintenance wash 

between runs or as needed. The machines are washed using Tween 20 (SigmaAldrich, catalog # P7949) mixed with Laboratory-grade water. The instructions 

provided by illumina to create your tween 20 wash solution is as follows: Add 5 ml of 

100% Tween 20 to 45ml laboratory grade water. These volumes result in 10% Tween 

20. Add 10% Tween 20 to 475 ml laboratory grade water. This results in a 0.5% 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 94 of 98

For the most current and official copy, check QMiS. 

Tween 20 wash solution. Mix solution well and add 6ml wash solution to each section 

of the wash tray, as well as 350 mls to the 500ml wash bottle. Repeat this each time 

for the three-step wash cycle. Some Illumina representatives will recommend 

replacing some of these washes with only laboratory grade water so remember to 

consult on this process locally. 

F. Which QA/QC tools should you always run? How do you know when you need to utilize 

additional tools? 

You should always run the GalaxyTrakr tool MicroRunQC. If the ST value of your 

isolate matches any of your internal controls or any of your labs, in the last six months, 

you need to run the SNP Pipeline. 

G. What should you do if you suspect an isolate is a result of a contamination event 

involving a laboratory control or another isolate? 

You should report to your supervisor and WGS Program Coordinators and initiate an 

issue in QMIS so that you can investigate and root causes analysis. 

H. What quality metrics should you look for during a sequencing run? 

Sequence Analysis Viewer and MicroRunQC results. 

Select Agents

A. What is a select agent? 

A select is a microorganism or chemical toxin capable of causing serious harm or 

death due to exposure through inhalation. 

B. Name four areas of training necessary before handling select agents. 

Physical containment, PPE, Biosafety Practices, Microbiological Characterization of 

Select Agents. 

C. Which agency regulates the use, possession and transfer of select agents capable of 

posing a serious public health threat? 

The CDC 

D. List the PPE required to work with select agents. 

Closed front gown or jump suit, double gloves, respirator or face mask, hair and shoe 

coverings. 

E. What work practices are to be in place when working in a Biological Safety Level (BSL) 

laboratory? For BSL-3? 

Laboratory access is limited or restricted. Personnel at increased risk for acquiring 

infection, or for whom infection would have serious consequences, are not allowed in 

the laboratory or animal rooms. Laboratory doors are kept closed when experiments 

are in process. Personnel wash their hands after handling microorganisms, when 

removing gloves, and before leaving the laboratory. Soap and disposable towels are 

readily found at sinks in the microbiology laboratories. No eating, drinking, chewing 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 95 of 98

For the most current and official copy, check QMiS. 

gum, smoking, handling medications, handling contact lenses, or applying cosmetics is 

permitted in the laboratory. All foodstuffs are stored outside of the laboratory. Mouth 

pipetting is prohibited. There is a program for handling and disposing of sharps using 

special sharps' containers. Work surfaces are decontaminated on completion of work 

and at the end of the day. This usually involves a system of various decontamination 

sprays, such as bleach, iodine, quaternium ammonium materials, amphyll, etc. Any 

splashes or spills of viable material are immediately cleaned up with disinfectants that 

are effective against the organism of concern. Contaminated equipment is to be 

decontaminated before it is sent for repair, sent for maintenance, placed in surplus, or 

shipped out of the lab. When placing the equipment in surplus, the equipment is also 

labeled in accordance with the DHHS requirements for property management to show 

that the equipment is clean. 

All cultures, stocks, and other regulated wastes are autoclaved, or otherwise 

decontaminated, to destroy any viable organisms. All materials for autoclaving are 

placed in red (or otherwise equivalent) bags that are closed for transport out of the 

laboratory. These bags are set in leak-proof totes. Any materials to be decontaminated 

off-site are packaged in accordance with applicable local, state, and federal regulations 

beforehand. When working with organisms with more hazardous risk, consideration is 

given to chemically decontaminating the wastes within the laboratory. An insect and 

rodent control program are in place. All procedures are conducted in a manner to 

minimize aerosols. Consideration is given to the degree of hazard when loops are 

used in flames, centrifugation, opening screw-capped bottles and wet petri dish covers, 

use of a syringe, needle, and septum, streaking plates, pipetting, slide agglutination, 

etc. Good microbiological techniques, substitution of disposable equipment, and use of 

primary containment devices such as biosafety cabinets reduce the risk. Laboratory 

management establishes policies and procedures whereby personnel in the laboratory 

are informed of the potential hazards and meet special entry requirements, such as 

immunization. A biohazard sign is posted at the entrance to any laboratory where 

etiologic agents are used. This label identifies what etiologic agents are used, the 

biosafety level, any required immunizations, the responsible person, and their phone 

number, what PPE is worn in the laboratory, and any procedures for exiting the 

laboratory. Immunizations or tests are offered to personnel for the agents handled or 

potentially present in the lab. Baseline blood serum samples may be collected and 

stored, depending on what agents are handled. Biosafety procedures are incorporated 

into individual SOPs, or a Biosafety Manual is adopted or prepared for the local 

laboratory. Personnel are advised of special hazards and are instructed to read and 

follow instructions on practices and procedures. Laboratory and support personnel 

receive training on potential hazards associated with the work performed, the 

precautions to avoid exposure, and exposure evaluation procedures. Training is 

updated annually or when the procedure or policy changes. Substitution of glassware 

for plasticware is recommended. A high degree of caution is taken with any procedure 

using needles and syringes, or any other sharps. Sharps should be restricted 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 96 of 98

For the most current and official copy, check QMiS. 

whenever possible. Only needle-locking syringes or disposable syringes should be 

used for injections, etc. Needles should not be manipulated in any manner before 

disposal; they should be carefully placed into a conveniently located puncture-resistant 

container used for sharps disposal. Non-disposable sharps are to be placed in a hardwalled container for transport to a processing area for decontamination, preferably by 

autoclaving. Syringes that re-sheathe the needle, needleless systems, and other 

safety devices are used when possible. Never handle broken glassware directly by 

hand. Use a mechanical means, such as brush and dustpan or tongs to pick up broken 

glass pieces. Dispose of broken glass in special containers. Containers of broken 

glass, sharps, and contaminated needles are decontaminated prior to disposal, or are 

shipped off-site in accordance with local, state, or federal regulations. 

Any cultures, tissues, specimens of body fluids, or potentially infectious wastes are 

placed in a container with a cover that prevents leakage during collection, handling, 

processing, storage, transport, or shipping. All spills and accidents that result in overt 

exposures to infectious materials are immediately reported to the Designated 

Laboratory Official. Medical evaluation, surveillance, and treatment are provided as 

needed and written records are maintained in accordance with 29 CFR 1910.1020. 

Only animals involved with the work being performed are permitted in the laboratory. 

Correct personal protective equipment is worn in the laboratory. Disposable gloves 

are not reused, washed, or used when touching “clean” surfaces, such as the 

telephone, door handles, keyboards, etc. Properly maintained safety equipment, 

including biological safety cabinets (Class II) or other containment devices are used 

whenever procedures with a potential for creating infectious aerosols or splashes are 

conducted, or high concentrations or large volumes of infectious agents are used. 

High concentrations or large volumes of infectious material may be centrifuged in the 

open lab if sealed rotor heads or centrifuge safety cups are used and if these rotors or 

cups are only opened inside of the biosafety cabinet. All surfaces inside the laboratory 

are readily accessible for cleaning and can easily be decontaminated. No carpets or 

rugs are used in the laboratory. The biological safety cabinet is located where 

fluctuations of the room supply, and exhaust air do not cause the cabinet to operate 

outside its parameters for containment. The cabinet is located away from doors, from 

windows that can be opened, from heavily traveled laboratory areas, and from other 

potentially disruptive equipment so as to maintain the biological safety cabinets' air 

flow parameters for containment. Eyewashes are readily found throughout the 

laboratory and are flushed weekly. Outside windows are fitted with screens. 

Illumination is provided without glare or reflections. 

BSL 3? 

All practices in the BSL-2 laboratory are followed in the BSL-3 laboratory. All 

procedures involving the manipulation of infectious materials are conducted within 

biological safety cabinets or other physical containment devices, or by personnel 

wearing correct personal protective clothing and equipment. BSL-3 laboratory has 

special engineering and design features, such as a double door access zone and 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 97 of 98

For the most current and official copy, check QMiS. 

sealed penetrations in the facility. The exhaust air from the laboratory room is 

discharged directly to the outdoors. The ventilation to the laboratory is balanced to 

provide directional airflow into the room. Access to the laboratory is restricted 

whenever work is in progress or the BSL-3 organism is present, and the recommended 

Standard Microbiological Practices, Special Practices, and Safety Equipment for BSL-3 

are rigorously followed. The Designated Laboratory Official strictly controls access to 

the laboratory. Access is restricted to only those persons whose presence is needed 

for program or support purpose. The director has the final responsibility for assessing 

each circumstance and determining who may enter or work in the laboratory. The 

Designated Laboratory Official establishes policies and procedures whereby only 

persons who have been advised of the potential biohazard, who meet any identified 

entry requirements (e.g. immunization), and who comply with all entry and exit 

procedures, enter the laboratory or animal rooms. Laboratory and support personnel 

receive training on the potential hazards associated with the work involved, the 

precautions to prevent exposures, and the exposure evaluation procedures. Personnel 

receive annual updates or additional training as needed for procedural changes. The 

Designated Laboratory Official is responsible for ensuring that, before working with 

organisms at BSL-3, all personnel demonstrate proficiency in standard microbiological 

practices and techniques, and in the practices and operations the local laboratory 

facility. This might include prior experience in handling pathogens or cell cultures, or a 

defined training program provided by the director or other competent scientist proficient 

in safe microbiological practices and techniques. All open manipulations involving 

infectious materials are conducted in biological safety cabinets or other physical 

containment devices within the containment module. No work in open vessels is 

conducted on the open bench. Clean up is facilitated by using plastic-backed paper 

toweling on non-perforated work surfaces within biological safety cabinets. 

Spills of infectious materials are decontaminated, contained, and cleaned up by 

knowledgeable professional staff, or others properly trained and equipped to work with 

concentrated infectious material. Spill procedures are developed and posted. 

Potentially contaminated equipment is decontaminated before removal from the facility 

for repair or maintenance or packaging for transport, in accordance with applicable 

regulations. Cultures, tissues, specimens of body fluids, or wastes are placed in a 

container that prevents leakage during collection, handling, processing, storage, 

transport, or shipping. All potentially contaminated waste materials (e.g. gloves, lab 

coats, usual waste materials) from laboratories are decontaminated before disposal or 

reuse. Decontamination should occur in the immediate area of the BSL-3 unit. 

Protective laboratory clothing such as solid-front or wrap-around gowns, scrub suits, or 

coveralls are worn by workers when in the laboratory. Protective clothing is not worn 

outside the laboratory. Reusable clothing is decontaminated before being laundered. 

Clothing is changed when overtly contaminated. Gloves are worn when handling 

infectious materials, infected animals, and when handling contaminated equipment. 

Frequent changing of gloves accompanied by hand washing is recommended. 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Sciences

Document Number: 

MAN-000055 

Revision #: 03 

Revised: 

11 Jul 2023 

Title: 

ORA Lab Manual Vol. IV Section 2 - Food and Feed Microbiology (IV-02) Page 98 of 98

For the most current and official copy, check QMiS. 

Disposable gloves are not reused. All manipulations of infectious materials, necropsy 

of infected animals, harvesting of tissues or fluids from infected animals or embryonate 

eggs, etc., are conducted in a Class II or Class III biological safety cabinet. When a 

procedure or process cannot be conducted within a biological safety cabinet, then 

correct combinations of personnel protective equipment (e.g., respirators, face shields) 

and physical containment devices (e.g. centrifuge safety cups or sealed rotors) are 

used. Respiratory and face protections are used when in rooms containing infected 

animals 

The laboratory is separated from areas that are open to unrestricted traffic flow within 

the building, and access to the laboratory is restricted. Passage through a series of two 

self-closing doors is the basic requirement for entry into the laboratory from access 

corridors. Doors are lockable. A clothes change room may be included in the 

passageway. Each laboratory room contains a sink for hand washing. The sink is 

hands-free or automatically operated and is located near the room exit door. The 

interior surfaces of walls, floors, and ceilings of areas where BSL-3 agents are handled 

are constructed for easy cleaning and decontamination. Seams, if present, are sealed. 

Walls, ceilings, and floors should be smooth, impermeable to liquids and resistant to 

the chemicals and disinfectants normally used in the laboratory. Floors should be 

monolithic and slip-resistant. Consideration should be given to the use of covered floor 

coverings. Penetrations in floors, walls, and ceiling surfaces are sealed or capable of 

being sealed to facilitate decontamination. Openings such as around ducts and the 

spaces between doors and frames are capable of being sealed to facilitate 

decontamination. A method for decontaminating all laboratory wastes is found in the 

facility and utilized, preferably within the laboratory (i.e., autoclave, chemical 

disinfection, incineration, or other approved decontamination method). Consideration 

should be given to means of decontaminating equipment. If waste is transported out of 

the laboratory, it should be properly sealed and not transported in public corridors. 

Consideration is given to safe transport of samples potentially contaminated with BSL3 organisms into the BSL-3 suite. All such samples should be contained in proper 

packaging to meet regulatory criteria and should not be opened except inside a Class 

II or III biosafety cabinet. 

F. Does the laboratory need to register with the CDC when working with regulated select 

agents? 

If the lab receives or ships any of these agents for analysis or research or participates 

in a collaborative study, then the lab must be registered with CDC to ship or receive 

any select agents.