Center for Meat Process Validation 

Annual Report on Research Supporting Critical Limit Choices 

November 5, 2004 

University of Wisconsin-Madison 

Dennis R. Buege and Steven C. Ingham 

In 2003-2004, we completed several research projects providing scientific 

evidence for Critical Limit choices by meat and poultry processors. The results of 

these studies are summarized in the table below. The table also identifies the 

attached report containing detailed information on each study. In addition, 

several of the studies have been accepted for publication by a peer-reviewed 

scientific journal. The citation for each of these articles is also listed in the table. 

To obtain a copy of an article, please contact Steve Ingham at 608-265-4801 or 

scingham@wisc.edu. The research described in this report, along with 

information from ongoing research, will also be available on our website 

http://www.wisc.edu/foodsafety/meatresearch/ 

. 

1 
SUMMARY OF STUDIES COMPLETED TO-DATE 

Product/Step Pathogens of Concern Critical Limits Supported by Results Report Article CiHama/Cooking Staphylococcus aureus Raw meatb/ 

Warmest Step 

Escherichia coli O157:H7 Salmonella spp. 

Raw poultryc 

Warmest Step 

Escherichia coli O157:H7 

Salmonella spp. 

Pre-cooked Meatsd/ 

Warmest Step 

Escherichia coli O157:H7 

Salmonella spp. 

Head Cheese/ Formulation 

Listeria monocytogenes 

Internal temp. of 50-93°F for< 4 h, 93-115°F for <5 h, #1 (p.6) Journal o

 and 115 - 130°F for <5 h. Volume 6

Exposure to 41- 50°F for <8 h or exposure to 41-72°F #2 (p. 29) Journal o

for < 2 h; product chilled to < 41°F by end of exposure In Press 

time limit. 

Exposure to 41- 50°F for <8 h; product chilled to #2 (p. 30) Journal o

< 41°F by end of exposure time limit. In Press 

Exposure to 41- 50°F for <6 h or exposure to 41 - 70°F #3 (p. 54) None for < 5 h; product chilled to < 41°F by end of exposure 

 time limit. 

pH of 4.2 – 4.4 and refrigeration at <41°F #4 (p. 68) None 
Snack Sticks/ Listeria monocytogenes various pH, % water-phase salt and #5 (p. 75) Journal o

Processing water activity combinations (see Table below) In Press and Formulation Summer Listeria monocytogenes various pH, % water-phase salt #5 (p. 75) Journal oSausage/Processing

 and 

water activity combinations (see Table below) In Press 

and Formulation Beef Jerky/ Listeria monocytogenes various pH, % water-phase salt #5 (p. 75) Journal o

Processing

 and 

water activity combinations (see Table below) In Press 

and Formulation Pork Rind/ Listeria monocytogenes various pH, % water-phase salt #5 (p. 75) Journal o

Cracklin Processing and water activity combinations (see Table below) In Press 

and Formulation 
a

Ham must be pumped with brine to attain at least 2.3% sodium lactate, at least 0.8% sodium chloride (salt) 

and at least 200 ppm ingoing sodium nitrite. 

b

Ground beef, intact beef, intact pork, bratwurst, uncooked corned beef. 

c

Chicken white meat and chicken dark meat. 

d

Pre-cooked pork chops, turkey slices and ham slices in the “heat treated but not fully cooked, not shelfstable” HACCP category. 

4 
Combinations of pH, % water-phase salt, and water activity that inhibited growth of 

Listeria monocytogenes on ready-to-eat meat products. For detailed information, please 

see report #5, starting on page 73. 

Product Category pH % water-phase salta water activity 

Beef/Venison Snack Sticks 4.8 7.6 0.91 

5.0 5.6 0.95 

5.0 5.9 0.93 

Summer Sausage and 4.7 3.9 0.96 

Related Sausage Products 4.9 5.2 0.95 

4.8 5.0 0.96 

5.2 6.5 0.95 

5.3 4.5 0.96 

Beef Jerky 5.6 14.4 0.75 

Pork Rinds/Cracklins 6.0 56.9 0.29 

6.1 60.7 0.27 

6.1 69.3 0.27 

6.7 69.2 0.28 

a

 Also referred to as brine concentration. To calculate, divide the % salt by the sum of % salt and 

% water. Multiply the answer by 100. 

5 
Report #1: Evaluating S. aureus growth potential in slow-cooked hams 

Modified version of article published in the Journal of Food Protection, Volume 

67, pages 1512-1516 

A Research Note 

Evaluation of Staphylococcus aureus Growth Potential in Ham During a 

Slow-Cooking Process: Use of Predictions Derived From the USDA-PMP 

6.1 Predictive Model and an Inoculation Study 

Steven C. Ingham*1

, Jill A. Losinski1, Brenda K. Dropp1, Laura L. Vivio1, 

and Dennis R. Buege2 

University of Wisconsin-Madison 

1

 Department of Food Science 

2

Department of Animal Sciences 

*Corresponding author 

University of Wisconsin-Madison, Department of Food Science 

1605 Linden Drive, Madison, WI 53706-1565 

Phone 608-265-4801 Fax 608-262-6872 

e-mail scingham@wisc.edu 

6 
ABSTRACT 

The United States Department of Agriculture (USDA) has cautioned against 

slow-cooking of meat such that the interior temperature increases from 50°F 

(10°C to 130°F (54.4°C) in > 6 h. During a commercial ham-smoking 

process, the ham cold-point is typically between 50 and 130°F (10 and 

54.4°C) for 13 h, but is later heated enough to kill vegetative pathogenic 

bacteria. Thus, production of heat-stable staphylococcal enterotoxin is the 

primary biological hazard. For this study, uncooked surface and uncooked 

ground interior ham were inoculated with a three-strain Staphylococcus 

aureus mixture, exposed to simulated surface and interior slow-cook 

conditions, respectively, and analyzed periodically using the Baird-Parker 

agar (B-P) and 3MTM PetrifilmTM Staph Express (PF-SE) count plate methods. 

For the surface and interior conditions, respectively, S. aureus numbers 

increased by no more than 0.1 and 0.7 log units. Predictions derived from 

actual time/temperature data and S. aureus growth values from a computergenerated model (PMP 6.1, USDA) were for 2.7 (ham surface) and 9.9 – 10.5 

(ham interior) generations of S. aureus growth, indicating that use of modelderived growth values would not falsely indicate safe slow-cooking of ham. 

The B-P method recovered significantly (P < 0.05) greater numbers of S. 

aureus than the PF-SE method. For hams pumped with brine to initially attain: 

1) 18% (w/w) weight gain, 2) > 2.3% sodium lactate, 3) > 0.8% sodium 

chloride, and 4) 200 ppm sodium nitrite, slow-cooking critical limits of < 4 h 

between 50 and 93°F (10°C and 34°C), <5 h between 93 and 115°F (34°C 

7 
- and 46°C), and < 5 h between 115 and 130°F (46 and 54.4°C) could be 

considered adequate to ensure safety. 

8 
-

INTRODUCTION In 1999, the United States Department of Agriculture (USDA) 

issued microbiological performance standards to be followed in the cooking of 

beef and poultry products (3). USDA has also published a compilation of product 

interior time/temperature combinations considered validated to meet the 

performance standards (4). This compilation, known in the meat and poultry 

industry as Appendix A, is legally applicable to certain beef and poultry products, 

but has also been used as a guide for cooking pork products. Appendix A 

cautions processors against slow-cooking processes in which the interior of the 

product increases from 50°F (10°C) to 130°F (54.4°C) in greater than six hours. 

Although slow-cooking procedures are not expressly forbidden, the onus for 

safety validation is on processors using them. Under the mandatory Hazard 

Analysis Critical Control Point (HACCP) system, validated time/temperature 

parameters would likely serve as critical limits for the Critical Control Point of 

cooking in a slow-cooking process. Thus, studies are needed to examine the 

safety of slow-cooking procedures and possibly validate critical limits for their use 

under the HACCP system. 

One common industry slow-cooking procedure is the smoking of large bone-in 

and boneless hams. During this process, the ham interior is eventually exposed 

to lethal temperatures (> 130°F or 54.4°C) for times far longer than necessary to 

meet the performance standards for destruction of Salmonella spp. (4). There is 

little historical evidence to suggest that growth of spore-forming pathogenic 

bacteria during slow-cooking of ham is a significant hazard. For example, out of 

9 
680 reported foodborne illness outbreaks caused by pathogenic bacteria in 1999­

2001, only 19 and 76 were caused by Bacillus cereus and Clostridium 

perfringens, respectively (8). Of these few outbreaks, pork was a potential 

vehicle in only two C. perfringens outbreaks and ham was never a potential 

vehicle. Furthermore, the presence of added sodium chloride and relatively high 

concentrations of sodium nitrite appears likely to minimize the risk of germination, 

growth, and toxigenesis by Bacillus cereus (6,16) and germination, growth, and 

sporulation-linked toxigenesis by Clostridium perfringens (13,15,17). In contrast, 

there is an association between ham and outbreaks of Staphylococcus aureus 

intoxication. During the same 3-year period cited above, 63 of the 680 outbreaks 

were caused by S. aureus, with 11 involving ham (8). Thus, the most significant 

biological hazard in the slow-cooking of hams is production of heat-stable 

enterotoxin by Staphylococcus aureus. Intoxication resulting from growth and 

enterotoxin production by S. aureus in foods is estimated to account for 1.3% of 

United States foodborne illness cases, but a high proportion of cases are 

believed to be sporadic and unreported (14). The intoxication rarely results in 

hospitalization, and is even less likely to cause death. 

There are two plausible ways in which hams could be contaminated with S. 

aureus before cooking. First the surface of the ham could be contaminated as a 

result of slaughter, fabrication, or handling. A USDA baseline microbiological 

survey of 2,112 market hogs in 1995-1996 found that 16.0% of the carcasses 

were contaminated with S. aureus, with a mean level of 1.92 log CFU per square 

10 
cm for samples testing positive (2). Humans, including food handlers (1,10,12), 

have been identified as frequent carriers of S. aureus, and the ham surface could 

become contaminated with this organism during processing and handling (7). 

The second possible route of contamination is via the brine solution that is 

pumped throughout the ham using long needles. If the brine solution or pumping 

apparatus is contaminated with S. aureus, the organism could be introduced into 

the ham interior. Likewise, S. aureus cells on the ham surface could be carried 

into the muscle interior by the needles during pumping. In the process examined 

in the present study, the cure solution is prepared and refrigerated prior to 

pumping the ham. The present study considered potential S. aureus 

contamination of both the ham surface and interior. 

An initial evaluation of the potential for S. aureus growth during slow-cooking 

is possible using actual product time/temperature data and S. aureus growth 

values from a computer-generated predictive model. The USDA Agricultural 

Research Service has developed such models, collectively known as PMP 6.1 

(USDA-ARS, Eastern Regional Research Center, Wyndmoor, PA; note that a 

more recent version of the models is now available via the internet), for several 

food-borne pathogens. Although the models are based on data from studies 

using isothermal conditions in a laboratory medium, the senior authors have 

frequently used these models as a primary tool in evaluating meat/poultry 

processing critical limits and deviations. However, computer-generated 

predictive models are not acceptable as the sole means of validating critical 

11 
limits. Other methods, such as inoculation studies, are needed to fully validate 

critical limits. 

The objectives of the present study were 1) to evaluate a typical ham slowcooking procedure for S. aureus growth potential using S. aureus growth values 

from the PMP 6.1 model and an inoculation study, and 2) if the slow-cooking 

procedure was determined to be safe, to recommend potential critical limits for 

validation. 

MATERIALS AND METHODS 

Ham Processing Parameters. At a commercial meat processing facility, 

hams were pumped at a target level of 18% using (for 1565 lb. or 709 kg of 

pumped hams) a mix of 208.8 lb. (94.6 kg) water, 44.8 lb. (20.3 kg) sodium 

lactate, 16.3 lb. (7.4 kg) sodium chloride, 16.8 lb (7.6 kg) sucrose, 5.3 lb. (2.4 kg) 

of a commercial curing salt containing 6.25% sodium nitrite, 14.1 oz. (0.4 kg) 

sodium ascorbate, and proprietary amounts of phosphates and seasonings. 

Three pumped bone-in hams, weighing between 13.2 and 16.5 lb (6 and 7.5 kg), 

were shipped in refrigerated insulated containers to the laboratory immediately 

after pumping. 

Preparation of the Ham for Laboratory Studies. Each ham was stored 

at 41°F (5°C) until prepared for laboratory studies. To prepare samples, ham 

was placed on a surface previously treated with 70% ethanol and a flame12 
sterilized knife was used to slice off a 0.5 in (1.2 cm) exterior layer. The exterior 

layer was cut into 2 in x 2 in x 0.5 in (5 cm x 5 cm x 1.2 cm) standardized pieces, 

and placed in sterile petri plates for the exterior inoculation study. Next, sections 

of ham muscle were aseptically removed to reach the center. Interior muscle 

was aseptically removed and ground using a meat grinder with a plate having 

0.016 in / 4.0 mm holes (Univex, Model MG8912, Salem, NH) and previously 

treated with 70% ethanol. Ground interior meat (0.18 ounce / 5 g) was 

transferred to each of several sterile 0.85 oz (25ml) plastic tubes (Falcon brand, 

Fisher Scientific, Itasca, IL). Tubes were manually shaken to force meat to the 

bottom of the tube and minimize air pockets. All samples were stored at 41°F 

(5°C) until used. 

Inoculum Preparation. A cocktail was made using three strains of 

Staphylococcus aureus. Strains ATCC 25923 and ATCC 12600 were obtained 

from the American Type Culture Collection (Manassas, VA), and FRI 1007 was 

provided by Dr. Amy Wong (Food Research Institute, University of WisconsinMadison). Stock cultures were maintained at -4°F (-20°C) in Brain Heart Infusion 

Broth (BHIB; Difco, Becton-Dickinson, Mansfield, MA) containing 10% (w/v) 

glycerol (Fisher). Working cultures, maintained at 40°F (4°C) on Brain Heart 

Infusion Agar (BHIA; Difco) plates, were prepared monthly from frozen stock. To 

obtain working cultures, each strain was cultured twice at 95°F (35°C) in BHIB, 

streaked to a BHIA plate, incubated for 18-24 h at 95°F (35°C), and observed for 

culture purity. For each experiment, fresh cultures were prepared by transferring 

13 
a loopful of growth from the working culture plate to 0.3 oz (9 ml) BHIB and the 

inoculated tubes were incubated at 95°F (35°C) for 20-24 h. Cocktails were 

prepared by vortex-mixing each culture, combining the cultures into a 1.7 oz (50 

ml) conical tube (Falcon brand, Fisher) and centrifuging at 5,000 x g for 10 

minutes. The supernatant was decanted and cells were re-suspended to the 

original volume in Butterfield’s phosphate diluent (BPD; Nelson Jameson, 

Marshfield, WI). The cocktail was then serially diluted in BPD, and plated on 

Baird Parker Agar (B-P; Difco) and 3MTM PetrifilmTM Staph Express (PF-SE; 3M 

Microbiology, St. Paul, MN) for initial enumeration. The PF-SE plates were 

incubated at 95°F (35°C) for 24 h and the B-P plates were incubated for 48 h at 

the same temperature. If only red-to-purple colonies were observed on a PF-SE 

plate after 24 h, they were counted as presumptive S. aureus. If more than one 

type of colony was observed, a thermonuclease disk was applied to the PF-SE 

plate, and plates were re-incubated at 95°F (35°C) for 1 – 3 h. Colonies 

displaying thermonuclease activity, as indicated by a pink halo on the 

thermonuclease disk after the second incubation period, were counted as 

presumptive S. aureus. Circular, smooth, convex, and gray to jet-black colonies 

surrounded by an opaque zone on B-P were counted as presumptive S. aureus. 

Inoculation and Simulation of Ham Slow-Cooking Conditions. 

Surface ham pieces were inoculated with 0.003 oz (0.1ml) of a 1:1000 dilution 

(BPD) of the S. aureus cocktail. The inoculum was spread evenly over the meat 

surface using a sterile bent plastic spreader (Daigger, Vernon Hills, IL). Each 

14 
petri plate (no lid) containing the inoculated ham exterior piece was placed in a 

72°F (22°C) incubator. To simulate time/temperature data obtained for the 

commercial process of interest, the incubator temperature was increased after 30 

minutes to 99°F (37°C), followed by another increase to 117°F (47°C) after 1.5 h. 

This temperature was maintained for an additional 2.0 h. The ground interior 

ham in each tube was inoculated with 0.003 oz (0.1ml) of S. aureus cocktail 

(diluted 1:1000 in BPD) by using a pipette tip to insert inoculum in the center of 

the ground meat. To simulate time/temperature data obtained for the commercial 

process of interest, the samples remained at room temperature for 2 h before 

being placed in a 80°F (26.7°C) water bath (Precision Reciprocal Shaking Bath; 

Precision Scientific, Chicago, IL). Subsequent increases in temperature occurred 

hourly. To monitor temperature, a calibrated thermometer was placed in the 

center of 0.18 oz (5 g) of uninoculated ground ham in the same type of tube used 

for the inoculated ham. This tube was placed in the water bath amongst the 

other tubes. Both experiments were done in triplicate (one trial per ham) with 

duplicate samples analyzed at each sampling time in each trial. 

Enumeration of Inoculum Organisms Before and During SlowCooking. To evaluate survival of S. aureus during the slow-cooking ham 

procedure, samples were analyzed at periodic intervals. Each ham surface piece 

was placed in a sample bag, and 3.35 oz (99ml) BPD was added before 

stomaching for 2 minutes on medium using a Stomacher 400 lab blender 

(Fisher). To analyze each ham interior sample, ground ham was aseptically 

15 
removed from the Falcon tube to a sample bag, weighed, and BPD was added to 

make a 1:10 dilution. Samples were stomached for 2 minutes on medium using 

a Stomacher 400 lab blender. For both interior and exterior samples, 

subsequent dilutions were made in BPD and plated to B-P and PF-SE. The 

initial dilution was transferred to three B-P plates (0.01, 0.01 and 0.013 oz / 0.3, 

0.3, and 0.4 ml) and one PF-SE plate. All subsequent dilutions were transferred 

to one plate each of B-P and PF-SE. Plates were incubated as previously 

described, and typical colonies were counted and log CFU was calculated for 

each sample. To confirm that colonies counted were S. aureus, one presumptive 

colony from each plating method from each trial was further tested for cell 

morphology, gram reaction, and coagulase activity. Throughout the study, all 

presumptive colonies were confirmed as S. aureus. 

Deriving Predictions from S. aureus Growth Values from the PMP 6.1 

Model. For each 0.5 – 2 h interval of the ham cooking simulation, the S. aureus 

growth values from the PMP 6.1 predictive growth model were used to predict 

the proportion of S. aureus lag phase that would elapse or the number of 

generations of S. aureus growth that would occur. To obtain the growth values, 

the following environmental conditions were used in the model: aerobic growth 

(chosen because aerobic growth is more rapid than anaerobic), pH 6.5 (typical 

pre-pumping value for hams; curing causes a drop of about 0.2 pH units), 2.5% 

water-phase sodium chloride (calculated from “worst case” values of 1.8% 

sodium chloride and 68% moisture), and 150 ppm added sodium nitrite (highest 

16 
value allowed in the model). The model covers the temperature range of 50 to 

108°F (10°C to 42°C). For each time interval with temperatures below the 

optimum for S. aureus growth (93°F / 34°C), the maximum temperature for the 

interval was assumed to have occurred for the entire interval. For time intervals 

between 93°F (34°C) and the maximum temperature for S. aureus enterotoxin 

production (115°F / 46°C; 12) the lowest growth temperature in the interval was 

assumed to have occurred for the entire interval. It was assumed that S. aureus 

was initially in lag phase (plausible since both the ham and the cure solution are 

refrigerated),and that the lag time at each given temperature during heating was 

the same as for the corresponding temperature during an isothermal experiment. 

Based on these assumptions, the proportion of lag phase elapsing during each 

time interval was obtained from the model and the cumulative proportion of lag 

phase was calculated (see Table 1). Similarly, when the entire lag phase had 

elapsed, the number of growth generations predicted during each time interval 

was determined from the corresponding isothermal model information and the 

cumulative number of growth generations was calculated. 

Statistical Analyses. For the ham exterior data, mean log CFU (CFU) 

per piece of meat was calculated for each sampling time, along with standard 

deviation. The mean, and standard deviation of log CFU/g, were calculated for 

the ground interior ham for each sampling time. For each experiment, the paired 

t-test (release 12, Minitab, Inc., State College, PA) was done to determine if a 

17 
significant (P < 0.05) difference existed between the B-P and PF-SE enumeration 

methods. 

RESULTS AND DISCUSSION 

In both the ham surface and ham interior experiments, there was a small, but 

statistically significant (P < 0.05) difference between the two methods used for S. 

aureus enumeration. On average, the B-P method recovered slightly more cells 

than the PF-SE method. A previous study of inoculated smoked fish, Mozzarella 

cheese, and Parmesan cheese found a similar difference between the two 

methods for Mozzarella cheese samples stored at 40°F (4°C) for 28 and 42 days 

(11). However, there was no difference between the B-P and PF-SE methods for 

Mozzarella cheese samples stored 0 or 14 days, or for the Parmesan cheese 

and smoked fish. It is possible that some environmentally stressed S. aureus 

cells are unable to grow on the PF-SE plate, causing the difference observed in 

the present study. 

Contamination of the ham interior with S. aureus is less likely to occur than 

surface contamination. However, interior contamination poses a greater potential 

food safety hazard because the ham interior heats much more slowly than the 

surface. Predictions made from S. aureus growth values from the PMP 6.1 

computer-generated model were for 9.9 to 10.5 generations, roughly 3 log units, 

of growth for S. aureus exposed to the temperature conditions in each of the 

three trials. This level of growth would result in enterotoxin production sufficient 

to cause illness, about 106

 cells, if S. aureus numbers prior to growth were in the 

18 
3 log range (5). The inoculation studies showed that the predictions based on S. 

aureus growth values from the PMP 6.1 model, as applied to this situation, were 

extremely conservative. Observed increases in S. aureus numbers did not 

exceed 0.7 log units (Table 2), between two and three generations of growth, for 

the period during which the ham was within the temperature range supporting S. 

aureus growth. It is likely that the ham composition was not as suitable for 

growth as the broth-based media used in obtaining data for the computergenerated model. Interestingly, during two of the three trials, S. aureus numbers 

began to decline during hour 14 of the experiment, suggesting that death of the 

organism begins between 127 and 130°F (52.5 and 54°C). This decrease 

accounted for the noticeably higher standard deviation observed for hour 14 data 

(Table 2). 

Surface contamination of ham with S. aureus is more likely to occur than 

interior contamination, but the ham surface is exposed to suitable growth 

temperatures for a much shorter time than the interior. Predictions based on S. 

aureus growth values from the PMP 6.1 model were for 2.7 generations of S. 

aureus growth under temperature conditions to which the ham surface was 

exposed. This level of growth would only result in production of illness-causing 

amounts of enterotoxin if initial numbers of S. aureus were extremely high. The 

inoculation studies showed no significant increase in S. aureus numbers during 

the heating period (Table 3). 

For hams having a composition similar to those used in this study, the 

inoculation study results clearly showed that slow-cooking critical limits for 

19 
product internal temperature of < 4 h between 50 and 93°F (10°C and 34°C), <5 

h between 93 and 115°F (34°C and 46°C), and < 5 h between 115 and 130°F (46 

and 54.4°C) will not allow growth of S. aureus to levels where enough toxin is 

produced to cause illness. Thus, the critical limits are safe and could be 

validated for use in HACCP plans. Key compositional levels that should be met 

for pumped hams cooked according to these critical limits would be a weight gain 

of 18% (w/w), > 2.3% sodium lactate, > 0.8% sodium chloride and 200 ppm 

sodium nitrite (the legal maximum). However, since brine has been shown to be 

a source of bacterial contamination (9), processors should frequently change the 

brine and clean and sanitize the brine container and pumping needles. Periodic 

microbiological testing is recommended to verify that the cure solution and 

pumping needles are not contaminated with high numbers of S. aureus. It is 

clear from our results that the S. aureus growth values from the PMP 6.1 model 

can be safely used to evaluate the safety of ham slow-cooking procedures, 

although such use of the values will greatly overestimate S. aureus growth. 

ACKNOWLEDGEMENTS This work was partially supported by the United 

States Department of Agriculture, Food Safety and Inspection Service, contract 

USDA-FSIS-27-2003. 

20 
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23 
Table 1. Use of Staphylococcus aureus growth values from the PMP 6.1 predictive model to estim

aureus growth during slow-cooking of ham. Growth parameters obtained for the following environm

aerobic growth, pH 6.5, 2.5% water-phase sodium chloride, and 150 ppm added sodium nitrite. Timdata used was based on actual commercial conditions. Time Length of Actual Assigned Lag Time % Lag Phase Generation TiElapsed Interval Temp. a Temp. From Model Calculatedb From Model 

Surface Runs 1-3 0.5 h 0.5 h 72 – 99 99 1.8 h 27.8 (27.8) 0.6 h 

1.0 h 0.5 h 99 99 1.8 h 27.8 (55.6) 0.6 h 1.5 h 0.5 h 99 - 117 99 1.8 h 27.8 (83.4) 0.6 h 

3.5 h 2.0 h 117 108 2.3 h 16.6 (100) 0.6 h Interior Run 1 2.0 h 2.0 h 48 – 73 73 5.8 h 34.5 (34.5) 1.8 h 

3.0 h 1.0 h 73 – 81 81 3.1 h 32.2 (66.8) 1.1 h 
4.0 h 1.0 h 81 – 93 93 1.9 h 33.2 (100) 0.7 h 

5.0 h 1.0 h 93 – 97 97 1.8 h -----d 0.6 h 

7.0 h 2.0 h 

97 

– 107 99 1.8 h ----- 0.6 h 

8.0 h 1.0 h 107 – 111 107 2.2 h ----- 0.6 h 10.0 h 2.0 h 111 – 118 108 2.3 h ----- 0.6 h 

Interior Run 2 2.0 h 2.0 h 43 – 70 70 8.2 h 24.3 (24.3) 2.3 h 3.0 h 1.0 h 70 – 82 82 2.8 h 35.7 (70.0) 1.0 h 4.0 h 1.0 h 82 – 91 91 1.9 h 30.0 (100) 0.7 h 5.0 h 1.0 h 

91 

– 96 96 1.8 h ----- 0.6 h 

7.0 h 2.0 h 

96 

– 106 99 1.8 h ----- 0.6 h 

8.0 h 1.0 h 106 – 111 106 2.1 h ----- 0.6 h 10.0 h 2.0 h 111 – 117 108 2.3 h ----- 0.6 h 
Interior Run 3 

2.0 h 2.0 h 50 – 69 69 9.1 h 22.0 (22.0) 2.5 h 3.0 h 1.0 h 69 – 81 81 3.0 h 33.3 (55.3) 1.1 h 4.0 h 1.0 h 81 – 90 90 2.0 h 44.7 (100) 0.7 h 5.0 h 1.0 h 

90 

– 95 95 1.8 h ----- 0.6 h 

7.0 h 2.0 h 

95 

– 104 99 1.8 h ----- 0.6 h 

8.0 h 1.0 h 104 – 109 104 2.0 h ----- 0.6 h 10.0 h 2.0 h 109 – 115 108 2.3 h ----- 0.6 h 

a Air temperature for surface studies; temperature of interior ground ham for interior studies. Value bValue is % of lag time for the given time interval with cumulative % of lag time in parentheses. cAbsence of value indicates that the calculated lag phase had not ended. When value is provided i

generations for given time interval with cumulative number of generations in parentheses. 

dNo value provided because lag phase was completed in preceding time interval. 
Table 2: Log of Staphylococcus aureus CFU/g in ground interior ham meat during 

simulation of a commercial slow-cooking process. Values are mean (n=3) and 

standard deviation (SD). S. aureus was enumerated by plating on Baird-Parker agar 

(B-P) and the 3MTM PetrifilmTM Staph Express Count plate (PF-SE). 

Time 

Target Actual Log CFU/g Log CFU/g 

Temperaturea Temperature B-P PF-SE 

T(h) (°C) Mean (SD) Mean (SD) Mean (SD) 

0 5.0 47 (3.8) 3.0 (0.1) 2.9 (0.1) 

2 21.1 71 (2.3) 3.0 (0) 2.8 (0) 

3 26.7 82 (0.5) 2.9 (0.1) 2.9 (0.1) 

4 32.2 91 (1.6) 3.0 (0.1) 2.9 (0.1) 

5 35.0 96 (1.1) 3.2 (0.5) 2.9 (0.3) 

6 37.8 101 (0.9) 2.9 (0.1) 3.1 (0.5) 

7 40.6 105 (1.4) 2.9 (0.1) 3.0 (0.2) 

8 43.3 111 (1.1) 2.9 (0.1) 2.9 (0.1) 

9 45.6 115 (0.9) 3.0 (0.2) 2.9 (0.2) 

10 46.7 116 (1.4) 3.4 (0.4) 3.4 (0.5) 

11 47.8 118 (1.1) 3.7 (0.7) 3.4 (0.7) 

12 49.4 120 (2.0) 3.4 (0.8) 3.0 (0.8) 

13 52.8 127 (0.5) 3.7 (1.1) 3.0 (1.1) 

14 54.4 131 (1.4) 2.8 (1.5) 2.0 (1.2) 

a Based on typical ham interior temperature during a commercial slow-cooking 

process. 

27 
Table 3: Log of Staphylococcus aureus CFU per exterior piece of ham during 

simulation of a commercial slow-cooking process. Values are mean (n=3) and 

standard deviation (SD). S. aureus was enumerated by plating on Baird-Parker 

agar (B-P) and the 3MTM PetrifilmTM Staph Express Count plate (PF-SE). 

Log Log 

Incubator 

Time CFU/piece CFU/piece 

Temperaturea 

B-P PF-SE 

(h) °F Mean (SD) Mean (SD) 

0 41 2.6 (0.1) 2.5 (0.1) 

0.5 99 2.7 (0.1) 2.6 (0.1) 

1 99 2.7 (0.1) 2.6 (0.1) 

1.5 117 2.7 (0.1) 2.6 (0.1) 

2 .0 117 2.4 (0.2) 2.3 (0.1) 

2.5 117 2.4 (0.1) 2.3 (0.2) 

3 (n=2) 117 2.3 (0.4) 2.2 (0.3) 

3.5b 117 2.5 (0.1 2.5 (0.3) 

a

 Based on typical smokehouse temperature in a commercial ham slow-cooking 

process. 

b

 Experiment ended after 3.5 h because smokehouse temperature exceeded 

130°F (54.4°C). 

28 
Report #2 Short-term temperature abuse of raw meats 

Modified version of article accepted for publication by the Journal of Food Safety 

Growth of Escherichia coli O157:H7 and Salmonella Serovars on Raw Beef, 

Pork, Chicken, Bratwurst, and Cured Corned Beef: 

Implications for HACCP Plan Critical limits 

Steven C. Ingham1*, Jill A. Losinski1

, Katie L. Becker1, and Dennis R. Buege2 

University of Wisconsin-Madison 

1

Department of Food Science 

2 Department of Animal Sciences 

* Corresponding author: 

1605 Linden Drive, Madison, WI 53706-1565 

Phone 608-265-4801, fax 608-262-6872, e-mail scingham@wisc.edu 

29 
ABSTRACT Small amounts (0.35 – 0.88 oz / 10 – 25 g; 0.98 – 3.22 in2

 / 6.3 – 

20.8 cm2

 inoculated area) of raw ground beef; intact beef, pork, and chicken 

(dark and white meat); bratwurst, and cured corned beef were inoculated with 

Salmonella serovars and E. coli O157:H7, refrigerated 24 h at 41°F (5°C), and 

then held either at 48 - 52°F / 9 - 11°C for up to 8 h or at room temperature (68 - 

76°F / 20 – 24°C) for up to 2h. Except for a 0.2 log CFU increase in Salmonella 

spp. in ground beef during 2 h at room temperature, pathogens did not grow. 

Results of trials with commercial amounts of beef, pork, chicken, ground beef, 

and bratwurst exposed to 50°F (10° C) for 8 h or 72°F (22°C) for 2 h also showed 

no pathogen growth. Potential critical limits for processing of previously 

refrigerated raw meat products are exposure temperatures between 41 and 50°F 

(5 and 10 °C) for not more than 8 hours or between 41 and 72°F (5°C and 22°C) 

for not more than 2 hours. 

30 
INTRODUCTION Under the United States Department of Agriculture (USDA) 

regulations mandating the Hazard Analysis Critical Control Point (HACCP) 

system for ensuring safe meat and poultry products (USDA, 1996), a Critical 

Control Point (CCP) is defined as “a point, step, or procedure in a food process 

at which control can be applied and, as a result, a food safety hazard can be 

prevented, eliminated, or reduced to acceptable levels”. The major CCP in 

HACCP plans for raw meat and poultry products is often the step in the process 

at which the product is warmest. For example, the stuffing step may be the CCP 

in a process for making uncooked bratwurst. At such a step, certain critical limits 

must be met to ensure control of significant hazards previously identified by the 

processor when doing a hazard analysis. Two significant microbiological 

hazards associated with raw meat and poultry products are Salmonella serovars 

and Escherichia coli O157:H7. Processors of raw products may have limited 

control over whether these pathogens are present in the raw meat they receive 

for processing. Therefore, processors must select a CCP and associated critical 

limits that will minimize the risk associated with contaminated raw ingredients. 

Regulatory officials expect processors of raw products to use CCP critical limits 

(time and temperature) that have been scientifically validated to not allow growth 

of Salmonella serovars and E. coli O157:H7. For processors of raw poultry 

products, the existing regulatory limit of 55°F (13°C) for maximum product 

temperature during processing (United States Department of Agriculture, 2003) 

can be used as a critical limit, or a more restrictive critical limit may be chosen. 

Although validation of critical limits ideally should involve in-plant microbiological 

31 
testing (Brashears, et al., 2002), wide-scale microbiological testing is not feasible 

for most very small processors. The objective of this study was to provide initial, 

laboratory-based evidence supporting simple critical limits that could be validated 

for use in raw meat and poultry HACCP plans. 

MATERIALS AND METHODS 

Preparation of Inoculum Although contamination of raw meat or poultry 

by a single pathogen strain is possible in a plant setting, the present study used 

multi-strain “cocktails” of Escherichia coli O157:H7 and Salmonella serovars to 

account for potential variation. The following Escherichia coli O157:H7 strains 

were used: ATCC 43894, 51657, 51658, and 43895 (obtained from American 

Type Culture Collection, Manassas, VA; the first three strains were originally from 

infected patients and the fourth was from ground beef implicated in an outbreak), 

and USDA-FSIS-380-94 (obtained from Dr. John Luchansky, Food Research 

Institute, University of Wisconsin-Madison; originally from salami implicated in an 

outbreak). Salmonella serovars used were S. Hadar S21, S. Typhimurium S9, S. 

Infantis S20, S. Enteritidis E40, S. Anatum S14, and S. Heidelberg S13. All of 

the salmonellae were obtained from Dr. Eric Johnson, Food Research Institute, 

University of Wisconsin-Madison and had been used in previous challenge 

studies. The original source was unknown for strains S21 and S20, while strains 

S9, S13, and S14 were originally isolated from samples submitted to the 

Wisconsin State Laboratory of Hygiene. Strain E40 was a chicken ovary isolate. 

Frozen stock cultures were maintained in Brain Heart Infusion broth (BHIB; Difco, 

32 
-

Becton Dickinson, Sparks, MD) with 10% (v/v) added glycerol (Fisher Scientific, 

Itasca, IL). Working cultures were prepared by growing each strain for two 

passages in BHIB and then streaking on Brain Heart Infusion Agar (BHIA; Difco). 

Following growth on BHIA for 24 h at 95°F (35°C), the working cultures were 

stored at 41°F (5°C). For each trial, a colony of each culture was transferred 

separately to 0.3 oz (9 ml) of BHIB and incubated for 24 h at 95°F (35°C), and 

then centrifuged at 8,000 x g for 10 minutes. Each culture was then resuspended in 0.3 oz (9 ml) of Butterfield’s Phosphate Diluent (BPD, NelsonJameson, Marshfield, WI) and all of the cultures for each genus were combined. 

For bratwurst and corned beef experiments, and trials with commercial amounts 

of products, the cultures for each genus were combined in a sterile 1.7 oz (50-ml) 

plastic tube, centrifuged, decanted, re-suspended to the original volume in BPD 

and then the resulting cocktails for the two genera were combined, with an 

overall population for each genus of 9.6 – 10.3 log CFU/oz (8.1 – 8.8 log 

CFU/ml). 

Meat and poultry products For trials with small amounts (0.35 – 0.88 oz 

/ 10 – 25 g; 0.98 – 3.22 in2

 / 6.3 – 20.8 cm2 inoculated area) of meat, ground beef 

(20% fat), boneless beef round steak, boneless center cut pork chops, chicken 

leg quarters (connected drumstick and thigh, up to 6% retained water), and 

boneless skinless chicken breasts were purchased at a local grocery store 

(separate purchases for each trial), transported to the laboratory within 15 

minutes, and refrigerated < 24 h until used. Uncooked bratwurst, from three 

33 
different manufacturers, was purchased frozen from the same local grocery 

store, transported to the laboratory and thawed under refrigeration. None of 

these products contained ingredients added for the purpose of inhibiting 

microbial growth. Uncooked, cured, corned beef briskets with garnish spices 

added (proprietary mix) were shipped overnight from a local processor in cooler 

boxes with ice packs. The corned beef represented three production lots, with 

each lot being studied in an independent trial. The corned beef was vacuumpackaged and had been cured with up to 30% of a mixture of water, salt, sodium 

phosphate, sugar, hydrolyzed corn protein, monosodium glutamate, sodium 

nitrite, sodium erythorbate, sodium nitrate, flavoring, and dextrose. Although 

some of these ingredients could potentially inhibit Salmonella and E. coli 

O157:H7, their addition was done primarily to obtain desired color and flavor, and 

to inhibit endospore germination and outgrowth. The corned beef was 

refrigerated 4 d before use. To prepare samples, products were divided or cut 

into smaller portions (0.35 – 0.88 oz / 10 – 25 g; 0.98 – 3.22 in2

 / 6.3 – 20.8 cm2 

inoculation area) and stored at 41°F (5°C) until inoculation. The ground beef was 

subdivided and shaped into small “mini-patties” (0.9 oz / 25 g), and the beef and 

pork were cut into small pieces (1 in x 1 in / 2.5 cm x 2.5 cm surface area, 0.5 in / 

1.2 cm thick). For chicken, the skin was removed from the chicken parts and 

small pieces (1 in x 1 in / 2.5 cm x 2.5 cm surface area, 0.5 in / 1.2 cm thick) 

were cut from the surface muscle. Bratwursts (4-5 links per lot) were cut into 16 

equal-sized pieces. The pieces were left intact for external (casing surface) 

inoculation. For internal inoculation, each link was cut in half longitudinally, and 

34 
then the resulting half-links were subdivided into 16 equal-sized pieces. The 

outer layer of each corned beef brisket was aseptically removed and cut into 3 in 

x 3 in x 0.5 in thick (6.3 cm x 6.3 cm x 0.6 cm thick) pieces. 

For trials with commercial amounts of meat products, two beef rounds 

(average weight 58.3 lb / 26.4 kg), two beef briskets (average weight 8. 8 lb / 4.0 

kg), two boneless pork loins (average weight 9.3 lb / 4.2 kg), twelve boneless 

skinless chicken breasts (average weight 1.3 lb / 615 g), six 3.1 lb / 1.4 kg chubs 

of ground beef round, and 18 fresh pork bratwursts with natural casing (average 

weight 0.2 lb / 100 g) were purchased from local grocery stores and transported 

within 15 minutes to a controlled environment chamber at the University of 

Wisconsin-Madison where the products were stored at 41°F (5°C). 

Inoculation of Meat Pieces Each mini-patty or piece of meat was 

inoculated with 0.003 oz (0.1 ml) of a 1:1,000 dilution (BPD) of the E. coli 

O157:H7 cocktail and 0.003 oz (0.1 ml) of a 1:1,000 dilution of the Salmonella 

spp. cocktail. For bratwurst and corned beef pieces, the inoculation was done 

with a 1:10 dilution of the combined two-genus cocktail, 0.003 oz (0.1 ml) for 

bratwurst and 0.01 oz (0.3 ml) for corned beef. Each inoculated piece was 

aseptically transferred to a sterile sample bag and refrigerated for 24 h at 41°F 

(5°C) to ensure that inoculum cells were not actively reproducing, thus simulating 

the situation on meat shipped and stored under refrigeration. After this 24 h 

refrigeration period, enumeration of inoculum organisms was done for three 

35 
patties/pieces per storage treatment for each trial of each product type, as 

described below. 

In trials with commercial amounts of products, the package of product was 

aseptically opened, and the packaging material either folded back (beef, pork, 

ground beef) or removed (chicken, bratwurst). Products were inoculated with a 

1:10 dilution of the combined two-species cocktail as follows: each of two beef 

rounds was inoculated at four sites with 0.017 oz (0.5 ml) spread over each 4 in 

x 4 in (10 x 10 cm) site, each of two beef briskets was inoculated at four sites 

with 0.017 oz (0.5 ml) spread over each 3 in x 3 in (7.5 x 7.5 cm) site, each of 

two boneless pork loins was inoculated at four sites with 0.017 oz (0.5 ml) spread 

over each 2.5 in x 3 in (6.3 x 7.5 cm) site, each of twelve boneless skinless 

chicken breasts was inoculated with 0.017 oz (0.5 ml) spread over one half of the 

exposed surface, each of two chubs of ground beef round was inoculated over 

the entire upper surface with 0.03 oz (1.0 ml), the exterior of each of four fresh 

pork bratwursts was inoculated with 0.008 oz (0.25 ml) spread over the entire 

upper surface and four bratwursts were sliced open and for each bratwurst 0.008 

oz (0.25 ml) was spread evenly inside the cut. After a 30-minute wait to allow 

bacterial attachment, the packaging material was placed back over the 

inoculated surface of beef, pork, and ground beef. Inoculated chicken breasts 

and bratwursts were individually placed into commercial zip-lock food storage 

bags. Inoculated meat products then remained in the 41°F (5°C) environmental 

chamber for 24 h until exposure to potential growth conditions. 

36 
-

Exposure of Inoculated Meat Products to Potential Growth 

Conditions After refrigeration, the inoculated mini-patties and small meat pieces 

were analyzed for initial inoculum levels or exposed to either room temperature 

(68 – 76°F / 20 - 24°C) for 1 h or 2 h, or 48 – 52°F (9-11°C, in a refrigerated 

incubator) for 2 , 4, 6, or 8 h. Because of the small amounts of meat/poultry 

studied, environment temperature was monitored, instead of product 

temperature, using a thermocouple probe (K-type) with an attached data-logger 

(Model SP-150, Dickson Instruments, Addison, IL). Inoculated commercial 

amounts of meat products were exposed to a temperature of either 50°F (10°C) 

for 8 hours or 72°F (22°C) for 2 hours. In trials with commercial amounts of 

products, the environmental chamber temperature was then returned to 41°F 

(5°C). Thermocouple probes (K-type) were inserted just under the surface of a 

beef round and bratwurst during the 50°F (10°C) and 72°F (22°C) trials. An 

attached data-logger recorded the time and temperature at 5-minute intervals. 

Enumeration of Inoculum Organisms After the mini-patties or small 

meat pieces had been exposed to refrigeration and/or the various 

temperature/time combinations, surviving pathogens were enumerated. 

Enumeration was done for three samples following each storage treatment in 

each trial. Initially, 3.3 oz (99 ml) of BPD was added to a sample bag and the 

contents were stomached for 2 minutes using a Stomacher 400 lab blender 

(Fisher Scientific, Itasca, IL). Subsequent dilutions were made in BPD and 

spread-plates were prepared (one plate per dilution) on Sorbitol MacConkey agar 

37 
(SMAC; Oxoid, Inc., Ogdensburg, NY) and XLD agar (Oxoid) for enumeration of 

E. coli O157:H7 and Salmonella serovars, respectively. Plates were incubated at 

95°F (35°C) for 24 h, typical colonies (white/colorless on SMAC, black on XLD) 

were counted and log CFU was calculated for each mini-patty or piece. To 

confirm that the colonies counted were the inoculum organisms, each of five 

typical colonies per plating medium (trials with ground beef, intact beef, pork, 

chicken) or one colony per trial (trials with bratwurst and corned beef) was 

transferred to BHIA and incubated for 24 h at 95°F (35°C). A presumptive E. coli 

O157:H7 colony from each BHIA plate was then tested for Gram reaction, cell 

morphology, oxidase reaction, presence of O157 antigen (DrySpot latex 

agglutination kit; Oxoid), and, for trials with ground beef, intact beef, pork, and 

chicken, appearance of growth on Levine’s EMB agar (Difco). A presumptive 

Salmonella colony from each BHIA plate was tested for Gram reaction, cell 

morphology, oxidase reaction, and biochemical characteristics (API 20E kit; 

bioMerieux, Inc., Hazelwood, MO). Throughout the study, all presumptive 

colonies were confirmed as the appropriate inoculum species. 

For trials with commercial amounts of product, samples were analyzed 

after 24 at 41°F (5°C) and then after 4 and 8 h exposure to 50°F (10°C) or after 1 

and 2 h exposure to 72°F (22°C); samples were also analyzed 24 h after the 

50°F (10°C) trials. Each sample was obtained by excising the inoculated area 

with a sanitized (70% ethanol) knife. The sample was transferred to a stomacher 

filter bag and analyzed as described above. Confirmation tests confirmed 

presumptive colonies as described above. 

38 
Statistical analysis For trials with small amounts (0.35 – 0.88 oz / 10 – 

25 g; 0.98 – 3.22 in2

 / 6.3 – 20.8 cm2 inoculated area) of meat, the log CFU/piece 

values for each sample after a given storage treatment in a trial were averaged. 

Then, the three resulting values (three trials) for a given storage treatment were 

averaged. The resulting value was compared to the value obtained after 24 h at 

41°F (5°C) using the two-sample t-test (Minitab, release 12.22, Minitab, Inc., 

State College, PA) with a significance level of 0.05. No statistical analysis was 

done for trials with commercial amounts of meat. 

RESULTS AND DISCUSSION 

Initially, small amounts of ground beef, intact beef, pork, chicken dark meat, and 

chicken white meat; cured corned beef, and bratwurst were inoculated with multistrain cocktails of Salmonella spp. and E. coli O157:H7. Small amounts of 

product were used because they would warm up much faster than the large 

amounts of meat used in meat processing plants, thus increasing the likelihood 

of pathogen growth. Samples were inoculated with approximately 5 log CFU of 

E. coli O157:H7 and Salmonella spp., refrigerated at 41°F (5°C) for 24 h (to be 

sure that the inoculum organisms were not reproducing, simulating their condition 

in previously refrigerated meat) and then exposed to either 48-52°F (9-11°C) for 

up to 8 h, or room temperature (68-76°F / 20-24°C) for up to 2 h. In these initial 

trials, changes in log CFU/piece were small after all storage treatments tested, 

ranging from an increase of 0.2 to a decrease of 0.2 (Table 1). No value 

39 
obtained after any storage treatment was significantly different (P < 0.05) from 

the initial value, with the exception of that for Salmonella on the ground beef 

mini-patties after 2 h at room temperature (panel A); however, the latter 0.2 

increase in log CFU/piece is probably not of practical significance. It was clear 

from these results that short-term increases in raw meat temperature would have 

little effect on numbers of E. coli O157:H7 and Salmonella serovars present on 

meat and poultry products. Interestingly, the results suggested that short-term 

exposure of poultry pieces to temperatures above the 55°F (13°C) regulatory limit 

would not result in dangerous pathogen growth. The lack of growth observed in 

laboratory experiments was consistent with that predicted for E. coli O157:H7 

and Salmonella by the United States Department of Agriculture, Agricultural 

Research Service PMP 6.1 computer-generated predictive model (USDA-ARS, 

Eastern Regional Research Center, Wyndmoor, PA). For a pH 6.5 broth system 

with 0.5% water-phase salt and no added sodium nitrite (least restrictive 

conditions in the model), the model predicted that E. coli O157:H7 would remain 

in lag phase for 54.0, 6.6, and 3.6 h at 50, 68, and 76°F (10, 20, and 24°C), 

respectively. Generation times for E. coli O157:H7 at these temperatures were 

predicted to be 5.3, 1.1 and 0.7 h, respectively. For Salmonella under the same 

broth culture conditions, the model predicted lag times of 63.4, 6.9, and 4.5 h and 

generation times of 9.4, 1.0 and 0.6 h, respectively. 

The surface temperature for small pieces of meat would rapidly increase 

to at or near the storage temperature, with the interior temperature also 

increasing rapidly. In intact-whole muscle product (beef, pork, and chicken 

40 
pieces), pathogenic bacteria are only expected to be on the surface, since the 

interior of muscle tissue is virtually free of microbes. The surface is the first part 

of the samples to warm or cool when exposed to changing temperatures. Within 

practical limits, temperature changes within intact muscle have little effect on 

surface microbial growth. Ground meat (mini-patties and bratwurst) represents a 

different case since microbes are distributed throughout, and the slowest-cooling 

part of the ground meat mass is the key area when evaluating microbial growth. 

Compared to the samples used in our study, larger masses of meat would take 

considerably longer to increase in temperature, and thus there would be a 

smaller likelihood of pathogen growth. In addition, though, large masses of 

ground meat would take a longer time to cool once they did increase in 

temperature. Ideally, meat and poultry processors should determine actual 

processing plant and product temperatures at various times during the 

processing day, over an extended time, to fully understand their product 

temperature history. Additionally, processors should obtain microbiological 

testing data for indigenous microorganisms, e.g. coliform count, Aerobic Plate 

Count, for use in validating critical limits. If no increase in numbers of these 

indigenous microbes occurs when the proposed critical limits are followed, then 

the critical limits could be considered fully validated. Such an approach was 

taken by Brashears et al. (2002) to validate a beef fabrication critical limit of 

processing room temperature at 57°F (14°C) or lower for no more than 4 hours. 

Results of subsequent trials with commercial amounts of meat (Table 2) 

confirmed the findings of trials with small meat pieces. There was no evidence of 

41 
growth on any of the products during exposure to either 50°F (10°C) for 8 hours 

or 72°F (22°C) for 2 hours. In 50°F (10°C) trials the temperatures just below the 

surface of the beef round (largest piece of meat) and the bratwurst (smallest 

piece) did not exceed 47°F (8.3°C) during 8 hours and were back to 41°F (5°C) 

within 5.5 and 0.5 hours, respectively, after the room was re-cooled. In 72°F 

(22°C) trials, the beef round temperature did not exceed 55°F (13°C) during the 2 

h exposure time, and had cooled to 41°F (5°C) in 1.5 h after the room was recooled. The bratwurst surface temperature rose to 66°F (19°C) during 2 h at 

72°F (22°C), but had cooled to 41°F (5°C) within 1.25 h after the room was recooled. Samples analyzed 24 h after the end of the 50°F (10°C) exposure 

showed no evidence of pathogen growth. These findings suggest that pathogen 

growth is unlikely in re-cooling raw meat and poultry products after short-term 

exposure to 50 – 72°F (10 - 22°C) temperatures. 

Given that 41°F (5°C) is widely regarded as a safe temperature for 

preventing growth of non-psychrotrophic pathogenic bacteria in potentially 

hazardous foods (United States Food & Drug Administration, 2001), critical limits 

for a specific HACCP plan could address the time that the relevant temperature 

(product or processing room) is above this temperature. With temperature and 

microbiological data in hand, processors could establish scientifically valid critical 

limits involving processing plant or product temperatures and times, and design 

their monitoring programs accordingly. Our results suggest that the following 

times and temperatures should be considered potential critical limits for 

preventing the growth of Salmonella spp. and E. coli O157:H7 on raw meat and 

42 
poultry: products can be exposed to temperatures between 41 and 50°F (5 and 

10 °C) for not more than 8 hours or to temperatures between 41 and 72°F (5°C 

and 22°C) for not more than 2 hours. The existing regulatory limit for raw poultry 

temperature during processing is supported by our results. Furthermore, our 

poultry results could be useful in evaluating poultry processing deviations 

involving short-term temperature elevation. 

ACKNOWLEDGEMENTS This work was partially funded by the United States 

Department of Agriculture, Food Safety & Inspection Service, contract USDAFIS-27-2003. 

REFERENCES 

1. BRASHEARS, M.M., E.S. DORMEDY, J.E. MANN, and D.E. BURSON. 

2002. Validation and optimization of chilling and holding temperature 

parameters as Critical Control Points in raw meat and poultry processing 

establishments. Dairy, Food and Environmental Sanitation 22, 246-251. 

2. UNITED STATES DEPARTMENT OF AGRICULTURE. 1996. Pathogen 

reduction; hazard analysis and critical control point (HACCP) systems. 

Final Rule. Federal Register 61 (144): 38805-38855. 

3. UNITED STATES DEPARTMENT OF AGRICULTURE. 2003. Poultry 

products inspection regulations – temperatures and chilling and freezing 

procedures. 9 CFR part 381.66. 

43 
4. UNITED STATES FOOD & DRUG ADMINISTRATION. 2001. Food Code 

2001, Recommendations of the United States Public Health Service, Food 

& Drug Administration. Section 3-501.16 and corresponding annex (pp. 

68, 309-310). National Technical Information Service, report number PB 

2002100819, Springfield, VA. 

44 
TABLE 1. 

LOG CFU/G OF SALMONELLA SEROVARS AND E. COLI O157:H7 ON SMALL 

AMOUNTS (0.35 – 0.88 oz / 10 – 25 g; 0.98 – 3.22 in2 / 6.3 – 20.8 cm2 INOCULATED 

AREA) OF MEAT AND POULTRY PRODUCTS STORED AT 41°F (5°C) FOR 24 H 

AND THEN AT EITHER 50°F (10°C) OR ROOM TEMPERATURE (RT; 68-76°F / 20-

24°C). PRODUCTS ARE GROUND BEEF MINI-PATTY (PANEL A), INTACT BEEF 

(B), INTACT PORK (C), CHICKEN WHITE MEAT (D), CHICKEN DARK MEAT 

(E), BRATWURST EXTERIOR (F), BRATWURST INTERIOR (G), AND CURED 

CORNED BEEF (H). 

A = ground beef mini-patty 

Storage Treatment Log CFU per piece A 

Conditions E. coli O157:H7 Salmonella serovars 

Initial (24 h, 41°F) 4.7 (0.1) 4.5 (0.1) 

1 h at RT 4.7 (0.1) 4.5 (0) 

2 h at RT 4.8 (0.1) 4.7 (0.1)B 

2 h at 50°F 4.7 (0) 4.5 (0) 

4 h at 50°F 4.7 (0.1) 4.5 (0) 

A

 VALUES ARE MEANS (N = 3) WITH STANDARD DEVIATIONS IN 

PARENTHESES. ABSENCE OF A SUPERSCRIPT INDICATES NO 

SIGNIFICANT DIFFERENCE (P > 0.05) FROM INITIAL VALUE). 

B VALUE IS SIGNIFICANTLY DIFFERENT (P < 0.05) FROM INITIAL VALUE. 

45 
B = beef piece 

Initial (24 h, 41°F) 4.6 (0.1) 4.6 (0.1) 

1 h at RT 4.7 (0.1) 4.6 (0.1) 

2 h at RT 4.8 (0.1) 4.6 (0.1) 

2 h at 50°F 4.7 (0) 4.5 (0.1) 

4 h at 50°F 4.8 (0.1) 4.6 (0) 

C = pork piece 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

Initial (24 h, 41°F) 4.6 (0) 4.5 (0.1) 

1 h at RT 4.5 (0) 4.5 (0.1) 

2 h at RT 4.5 (0.1) 4.5 (0.1) 

2 h at 50°F 4.6 (0.1) 4.5 (0.1) 

4 h at 50°F 4.5 (0.1) 4.5 (0.1) 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

46 
D = chicken white meat piece 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

Initial (24 h, 41°F) 4.7 (0.2) 4.3 (0.2) 

1 h at RT 4.6 (0.1) 4.2 (0.2) 

2 h at RT 4.7 (0.2) 4.3 (0.1) 

2 h at 50°F 4.8 (0.1) 4.3 (0.1) 

4 h at 50°F 4.7 (0.1) 4.2 (0.1) 

E = chicken dark meat piece 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

Initial (24 h, 41°F) 4.7 (0) 4.3 (0.1) 

1 h at RT 4.7 (0.2) 4.4 (0.1) 

2 h at RT 4.8 (0.1) 4.3 (0.1) 

2 h at 50°F 4.6 (0.1) 4.3 (0.1) 

4 h at 50°F 4.7 (0.1) 4.4 (0.1) 

47 
F = exterior of bratwurst 

Initial (24 h, 41°F) 5.3 (0.1) 5.2 (0.1) 

4 h at 50°F 5.1 (0.2) 5.0 (0.2) 

6 h at 50°F 5.2 (0.1) 5.1 (0.1) 

8 h at 50°F 5.2 (0.1) 5.1 (0.2) 

G = interior of bratwurst 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

Initial (24 h, 41°F) 5.5 (0.1) 5.4 (0.1) 

4 h at 50°F 5.5 (0) 5.4 (0.1) 

6 h at 50°F 5.5 (0.1) 5.3 (0.2) 

8 h at 50°F 5.5 (0.1) 5.3 (0) 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

48 
H = corned beef piece 

Initial (24 h, 41°F) 5.9 (0.1) 5.8 (0.1) 

4 h at 50°F 5.9 (0.1) 5.8 (0) 

6 h at 50°F 5.9 (0.2) 5.8 (0.1) 

8 h at 50°F 5.8 (0.2) 5.7 (0.2)) 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

49 
TABLE 2. 

LOG CFU/G OF SALMONELLA SEROVARS AND E. COLI O157:H7 ON 

COMMERCIAL AMOUNTS OF MEAT AND POULTRY PRODUCTS STORED 

AT 41°F (5°C) FOR 24 H AND THEN AT EITHER 50°F (10°C) OR ROOM 

TEMPERATURE (68-76°F / 22°C). PRODUCTS ARE BEEF ROUND (PANEL 

A), BEEF BRISKET (B), PORK LOIN (C), CHICKEN BREAST (D), GROUND 

BEEF (E), BRATWURST EXTERIOR (F), AND BRATWURST INTERIOR (G). 

A = beef round 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars. 

Initial (24 h, 41°F) 4.8 4.7 

1 h at 72°F 5.3 5.3 

2 h at 72°F 4.7 4.5 

Initial (24 h, 41°F) 5.3 5.1 

4 h at 50°F 5.5 5.4 

8 h at 50°F 5.3 5.1 

50 
B = beef brisket 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

Initial (24 h, 41°F) 5.1 5.1 

1 h at 72°F 4.7 4.5 

2 h at 72°F 4.6 4.4 

Initial (24 h, 41°F) 5.3 4.8 

4 h at 50°F 5.5 5.1 

8 h at 50°F 4.6 4.4 

C= Pork Loin 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

Initial (24 h, 41°F) 4.8 4.7 

1 h at 72°F 4.5 4.1 

2 h at 72°F 4.2 3.9 

Initial (24 h, 41°F) 4.7 4.7 

4 h at 50°F 4.8 4.6 

8 h at 50°F 4.5 4.4 

51 
D = Chicken breast 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

Initial (24 h, 41°F) 4.9 4.8 

1 h at 72°F 4.8 4.7 

2 h at 72°F 4.8 4.6 

Initial (24 h, 41°F) 4.6 4.1 

4 h at 50°F 4.6 4.3 

8 h at 50°F 4.9 4.7 

E = Ground beef 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

Initial (24 h, 41°F) 4.9 4.8 

1 h at 72°F 4.6 4.5 

2 h at 72°F 4.7 4.6 

Initial (24 h, 41°F) 4.6 5.3 

4 h at 50°F 5.2 4.9 

8 h at 50°F 5.0 4.8 

52 
F = Bratwurst exterior 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

Initial (24 h, 41°F) 4.6 4.3 

1 h at 72°F 4.8 4.4 

2 h at 72°F 4.7 4.4 

Initial (24 h, 41°F) 4.7 4.3 

4 h at 50°F 4.7 4.5 

8 h at 50°F 4.7 4.4 

G = Bratwurst Interior 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella serovars 

Initial (24 h, 41°F) 5.4 5.1 

1 h at 72°F 4.9 4.8 

2 h at 72°F 5.3 5.0 

Initial (24 h, 41°F) 5.5 5.2 

4 h at 50°F 5.1 5.1 

8 h at 50°F 5.0 5.2 

53 
Report #3 Short-term temperature abuse of cooked but not shelf-stable 

meat and poultry products 

Laboratory-Based Evidence Supporting Simple Critical Limits for Use with 

Cured Meat and Poultry Products in the “Heat Treated But Not Fully 

Cooked, Not Shelf-Stable” HACCP Category 

Steven C. Ingham1*, Jill A. Losinski1

, and Dennis R. Buege2 

University of Wisconsin-Madison 

1

Department of Food Science 

2 Department of Animal Sciences 

* Corresponding author: 

1605 Linden Drive, Madison, WI 53706-1565 

Phone 608-265-4801, fax 608-262-6872, e-mail scingham@wisc.edu 

54 
ABSTRACT Some processors receive or produce refrigerated fully cooked 

cured meat products, then subdivide and repackage the product into single 

portions for refrigerated or frozen distribution. During portioning and packaging, 

the product is not re-heated and, because the consumer is directed to fully cook 

the product, it is assigned to the United States Department of Agriculture (USDA) 

Hazard Analysis Critical Control Point (HACCP) plan category of “heat treated 

but not fully cooked, not shelf-stable”. In a HACCP plan for this category, a 

primary Critical Control Point is usually the step at which the product is warmest. 

This study was done to support the designation of simple Critical Limits that 

could be incorporated in HACCP plans for heat treated but not fully cooked, not 

shelf-stable products to prevent growth of Salmonella spp. and E. coli O157:H7. 

Single-portion cured pork chops, turkey slices, and ham slices, intended for recooking by the consumer, were inoculated with multi-strain cocktails of 

Salmonella spp. and E. coli O157:H7, refrigerated 24 h at 41°F (5°C), and then 

exposed to either 50°F (10°C) for 2, 4, or 6 h, or room temperature (70°F / 21°C) 

for 1, 3, or 5 h. The greatest increase in pathogen numbers during the shortterm exposure of pork chops to 50 – or 70°F (10 or 21°C) was 0.5 log CFU/piece 

with a statistically significant increase (P < 0.05) only observed after 5 h at 70°F 

(21°C) for Salmonella spp. (0.3 log CFU/piece increase). On ham and turkey 

slices, there was a 0.3 – 0.4 log CFU/piece increase in pathogen numbers when 

the products were first removed from refrigeration, which was probably 

attributable to recovery of injured cells. However, there was no significant 

increase in pathogen numbers thereafter, indicating that neither species was 

55 
growing. For full validation of Critical Limits, the individual processor should 

obtain actual plant and product temperature data, as well as microbiological data 

for their products. However the Critical Limits suggested by our results are that 

cured products in the heat treated but not fully cooked, not shelf-stable product 

category should not be between 41 and 50°F (5 and 10 °C) for more than 6 

hours, or between 41 and 70°F (5°C and 21°C) for more than 5 hours. 

INTRODUCTION Some processors receive or produce refrigerated fully 

cooked cured meat products, subdivide them into single portions, and repackage 

the products for refrigerated or frozen distribution. During this process, the 

products are not re-heated, and, because the consumer is directed to fully cook 

the products before consuming them, the products are assigned to the United 

States Department of Agriculture (USDA) Hazard Analysis Critical Control Point 

(HACCP) plan category of “heat treated but not fully cooked, not shelf-stable”. 

The major Critical Control Point (CCP) in HACCP plans for meat products in this 

category is usually the step in the process at which the product is warmest and 

conditions are most conducive for pathogen growth. For not fully cooked 

products, regulatory officials expect processors to use Critical Limits (time and 

temperature) for a CCP(s) that have been scientifically validated for preventing 

growth of Salmonella spp. and Escherichia coli O157:H7. Although validation of 

Critical Limits should ideally involve in-plant microbiological testing (1), widescale microbiological testing may not be feasible for all processors. In particular, 

challenge studies involving pathogenic bacteria should not be conducted in a 

56 
commercial meat processing facility. The objective of this study was to provide 

initial evidence, based on laboratory studies of pathogen growth, supporting 

simple Critical Limits that could be incorporated in the HACCP plan for cured 

meat and poultry products in the heat treated but not fully cooked, not shelfstable HACCP category. 

MATERIALS AND METHODS 

Overview Pork chops cut from cooked, cured pork loin and cured turkey 

and ham slices were inoculated with multi-strain cocktails of Salmonella spp. and 

E. coli O157:H7, refrigerated at 41°F (5°C) for 24 h (to be sure that the inoculum 

organisms were not actively growing and to simulate the situation in previously 

refrigerated meat) and then exposed to 50°F (10°C) for 2, 4, or 6 h or to room 

temperature (70°F / 21°C ) for 1, 3, or 5 h. Growth of the inoculum organisms 

during the short-term temperature increases was determined by plating on 

selective media. The experiment was performed in triplicate to obtain three 

independent trials. 

Pork Chops Pork chops cut from fully cooked and smoked cured pork 

loins were received via refrigerated truck from a processor. The pork loins had 

been cured with a mixture of water, salt, dextrose, sodium phosphates, sodium 

erythorbate, and sodium nitrite. The pork chops were approximately 0.7 in (1.8 

cm) thick and were vacuum-packaged prior to shipping. Upon receipt, the pork 

chops were stored at 41°F (5°C) until used. 

57 
Ham and Turkey Slices Individually vacuum-packaged cured ham and 

turkey slices (about 0.8 in / 2 cm thick) were received from a processor in 

insulated coolers containing ice packs. Other than ham and turkey, the 

ingredient statements for the two products were identical, reading “water, sodium 

lactate, salt, sugar, sodium phosphate, sodium ascorbate, sodium nitrite”. The 

ham and turkey slices were stored at 41°F (5°C) until used. 

Preparation of Inoculum Although contamination of raw meat or poultry 

by a single pathogen strain is possible in a plant setting, the present study used 

multi-strain “cocktails” of Escherichia coli O157:H7 and Salmonella spp. to 

account for potential strain-to-strain differences. The following Escherichia coli 

O157:H7 strains were used: ATCC 43894, 51657, 51658, and 43895 (obtained 

from American Type Culture Collection, Manassas, VA; the first three strains 

were originally from infected patients and the fourth was from ground beef 

implicated in an outbreak), and USDA-FSIS-380-94 (obtained from Dr. John 

Luchansky, Food Research Institute, University of Wisconsin-Madison; originally 

from salami implicated in an outbreak). Salmonella spp. strains used were S. 

hadar S21, S. typhimurium S9, S. infantis S20, S. enteritidis E40, S. anatum S14, 

and S. heidelberg S13. All of the salmonellae were obtained from Dr. Eric 

Johnson, Food Research Institute, University of Wisconsin-Madison. The 

original sources were unknown for strains S21 and S20, while strains S9, S13, 

and S14 were originally isolated at the Wisconsin State Laboratory of Hygiene. 

Strain E40 was a chicken ovary isolate. Frozen stock cultures were maintained 

58 
in Brain Heart Infusion (BHIB; Difco, Becton Dickinson, Sparks, MD) with 10% 

(v/v) added glycerol (Fisher Scientific, Itasca, IL). Working cultures were 

prepared by growing each strain for two passages in BHIB and then streaking on 

Brain Heart Infusion Agar (BHIA; Difco). Following growth on BHIA for 24 h at 

95°F (35°C), the working cultures were stored at 41°F (5°C). A colony of each 

strain was streaked on BHIA and grown for 24 h at 35°C, following which a 

colony of each culture was grown separately in 0.3 oz (9 ml) of BHIB for 24 h at 

95°F (35°C). All cultures for each species were combined, and then centrifuged 

at 5,000 x g for 10 minutes. Each resulting pellet was then re-suspended to 

original volume in Butterfield’s Phosphate Diluent (BPD, Nelson-Jameson, 

Marshfield, WI) and cocktails for the two species were combined. 

Inoculation of Meat Pieces Pork chops, ham slices, and turkey slices 

were laid on aluminum foil that had previously been treated with 70% (v/v) 

ethanol in a laminar flow bio-safety hood. Each pork chop was inoculated with 

0.3 ml of a 1:1,000 dilution (BPD) of the two-species cocktail which was then 

spread evenly using a sterile bent plastic rod (Daigger, Inc., Vernon Hills, IL). 

After inoculation, the pork chops, ham slices, and turkey slices were allowed to 

dry for 15 min., and then flipped over, and the inoculation procedure was 

repeated. Each inoculated piece was aseptically transferred to a vacuumpackaging bag (FoodSaver, Tilia, Inc., San Francisco, CA), vacuum-packaged, 

and refrigerated for 24 h at 41°F (5°C) to ensure that inoculum cells were not 

actively growing. After this 24 h refrigeration period, enumeration of inoculum 

59 
-

organisms was done for three pieces per storage treatment for each trial of each 

product type, as described below. 

Exposure of Inoculated Meat to Potential Growth Conditions After 

refrigeration, the inoculated pork chops, ham slices, and turkey slices were 

analyzed for initial inoculum levels or exposed to either 50°F / 10°C (in a 

refrigerated incubator) for 2, 4, or 6 h or room temperature (70°F / 21°C in an 

incubator) for 1, 3, or 5h. 

Enumeration of Inoculum Organisms After the pork chops, ham slices, 

and turkey slices had been exposed to refrigeration and/or the various 

temperature/time combinations, surviving pathogens were enumerated. 

Enumeration was done for three samples following each storage treatment in 

each trial. Each pork chop, ham slice, or turkey slice was aseptically removed 

from its package and a 1 in x 1 in 0.25 in thick (2.5 cm x 2.5 cm x 0.6 cm thick) 

piece of meat was excised from one side of the chop or slice and transferred to a 

sterile sample bag. Then, 3.3 oz (99 ml) of BPD was added to the sample bag 

and the contents were stomached at medium speed for 2 minutes using a 

Stomacher 400 lab blender (Fisher Scientific, Itasca, IL). Subsequent dilutions 

were made in BPD and spread-plated (one plate per dilution) on Sorbitol 

MacConkey agar (SMAC; Oxoid, Inc., Ogdensburg, NY) and XLD agar (Oxoid) 

for enumeration of E. coli O157:H7 and Salmonella spp., respectively. Plates 

were incubated at 95°F (35°C) for 24 h, typical colonies (white/colorless on 

60 
SMAC, black on XLD) were counted, and log CFU was calculated for each piece 

of pork chop. To confirm that colonies counted were the inoculum organisms, 

each of four typical colonies per plating medium was transferred to BHIA and 

incubated for 24 h at 95°F (35°C). Presumptive E. coli O157:H7 colonies were 

then tested for Gram reaction, cell morphology, oxidase reaction, and presence 

of O157 antigen (latex agglutination kit; Oxoid). Presumptive Salmonella spp. 

colonies from each BHIA plate were tested for Gram reaction, cell morphology, 

oxidase reaction, and biochemical characteristics (API 20E kit; bioMerieux, Inc., 

Hazelwood, MO). Throughout the study, the identity of all presumptive colonies 

was confirmed. 

Statistical analysis The log CFU/piece values for each sample after a 

given storage treatment in a trial were averaged (n = 3). Then, the three 

resulting values (three trials) for a given storage treatment were averaged. The 

resulting value was compared to the value obtained after 24 h at 41°F (5°C) 

using the two-sample t-test (Minitab, release 12.22, Minitab, Inc., State College, 

PA) with a significance level of 0.05. 

RESULTS AND DISCUSSION For pork chops, the changes in pathogen 

numbers, expressed in log CFU/piece, were small after all storage treatments 

tested, ranging from an increase of 0.3 to no change (Table 1). Except for 

Salmonella after 5 h at 70°F (21°C) and E. coli after 3 h at 70°F / 21°C (Table 1), 

no value obtained after any storage treatment was significantly different (P < 

61 
0.05) from the initial value. In the latter case, the 0.2 increase in log CFU/piece 

was probably not of practical importance because no significant increase in E. 

coli O157:H7 numbers was observed after a longer period of 70°F (21°C) storage 

(5 h). It is clear from these results that short-term increases in product 

temperature will have little effect on numbers of E. coli O157:H7 and Salmonella 

spp. present on the pork chops. 

For the ham and turkey slices, there was a statistically significant 

decrease in pathogen numbers during the 24 h of storage at 41°F (5°C) (Tables 

2 and 3). This decrease was 0.7 – 0.8 log CFU/piece. During the first 2 h at 

50°F (10°C) and the first 1 h at 70°F (21°C), pathogen numbers increased to 

levels 0.4 log CFU/piece lower than those before the 24 h 41°F (5°C) storage. 

Because pathogen numbers did not significantly increase during subsequent 

exposure to 50 or 70°F (10 or 21°C), we conclude that this initial increase was 

caused by recovery of cold-injured cells, rather than by actual growth in numbers. 

In the present study, we monitored the temperature of the meat storage 

environment, as well as the actual meat temperature. The meat temperature 

increased to near that of the environment. Thus monitoring room temperature for 

processing of relatively small products like refrigerated pork loin, ham slices, or 

turkey slices may be an appropriate approach for Critical Limit monitoring. 

Ideally, meat and poultry processors should determine actual processing plant 

and product temperatures at various times during the processing day, over an 

extended time, to fully understand their product temperature history. Additionally, 

processors should obtain microbiological testing data for indigenous 

62 
microorganisms, e.g. coliform count, Aerobic Plate Count, for use in validating 

Critical Limits. No increase in numbers of these indigenous microbes would 

support the validity of the proposed Critical Limits. Such an approach was taken 

by Brashears et al. (1) to validate a beef fabrication Critical Limit of processing 

room temperature at 57°F (14°C) or lower with meat exposure for no more than 4 

hours. 

Given that 41°F (5°C) is widely regarded as a safe temperature for 

preventing growth of non-psychrotrophic pathogenic bacteria in potentially 

hazardous foods (2), Critical Limits for a specific HACCP plan could address the 

time that the relevant temperature (product or processing room) is above 41°F 

(5°C). With temperature and microbiological data in hand, processors can 

establish scientifically valid Critical Limits involving processing plant or product 

temperature and times, and design their monitoring programs accordingly. As a 

guideline for developing these Critical Limits, our results suggest the following 

general Critical Limits for preventing the growth of Salmonella spp. and E. coli 

O157:H7 on pork chops, ham slices, or turkey slices in the heat treated but not 

fully cooked, not shelf-stable product category: products should not be between 

41 and 50°F (5 and 10 °C) for more than 6 hours, or between 41 and 70°F (5°C 

and 21°C) for more than 5 hours. 

ACKNOWLEDGEMENTS This project was funded by a grant from the United 

States Department of Agriculture, Food Safety and Inspection Service, contract 

number USDA-FSIS-27-2003. 

63 
REFERENCES 

1. Brashears, M.M., E.S. Dormedy, J.E. Mann, and D.E. Burson. 2002. 

Validation and optimization of chilling and holding temperature parameters 

as Critical Control Points in raw meat and poultry processing 

establishments. Dairy, Food and Environmental Sanitation 22: 246-251. 

2. Food & Drug Administration. 2001. Food Code 2001, Recommendations 

of the United States Public Health Service, Food & Drug Administration. 

Section 3-501.16 and corresponding annex (pp. 68, 309-310). National 

Technical Information Service, report number PB 2002100819, 

Springfield, VA. 

64 
Table 1. Mean (3 trials, each trial containing triplicate samples) log of Colony 

Forming Units (CFU) of Salmonella spp. and E. coli O157:H7 per pork chop 

sample after refrigeration (24 h at 41°F / 5°C) and subsequent storage at 50 or 

70°F (10 or 21°C). Values are means with standard deviations in parentheses. 

Absence of a superscript indicates no significant difference (P > 0.05) from initial 

value. 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella spp. 

Initial (24 h, 41°F) 4.6 (0.1) 4.4 (0.1) 

2 h at 50°F 4.8 (0.3) 4.6 (0.3) 

4 h at 50°F 4.8 (0.1) 4.6 (0.3) 

6 h at 50°F 4.7 (0.1) 4.6 (0.1) 

1 h at 70°F 4.6 (0.1) 4.5 (0.2) 

3 h at 70°F 4.8 (0.2) A 4.6 (0.3) 

5 h at 70°F 4.7 (0.1) 4.7 (0.1) A 

A

Value is significantly different (P < 0.05) from initial value. 

65 
Table 2. Mean (3 trials, each trial containing triplicate samples) log of Colony 

Forming Units (CFU) of Salmonella spp. and E. coli O157:H7 per ham slice 

sample at inoculation, after refrigeration (24 h at 41°F / 5°C), and after 

subsequent storage at 50 or 70°F (10 or 21°C). Values are means with standard 

deviations in parentheses. Absence of a superscript indicates no significant 

difference (P > 0.05) from initial value. 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella spp. 

At inoculation 5.2 (0.1) A 5.0 (0) A 

Initial (24 h, 41°F) 4.4 (0.1) 4.3 (0.2) 

2 h at 50°F 4.8 (0.1) A 4.6 (0.3) 

4 h at 50°F 4.8 (0.2) 4.7 (0.1) A 

6 h at 50°F 4.8 (0.3) 4.7 (0.1) A 

1 h at 70°F 4.8 (0.2) 4.7 (0.2) 

3 h at 70°F 4.9 (0.1) A 4.8 (0.1) A 

5 h at 70°F 4.8 (0.2) 4.7 (0.2) A 

A

Value is significantly different (P < 0.05) from initial value. 

66 
Table 3. Mean (3 trials, each trial containing triplicate samples) log of Colony 

Forming Units (CFU) of Salmonella spp. and E. coli O157:H7 per turkey slice 

sample at inoculation, after refrigeration (24 h at 41°F / 5°C), and after 

subsequent storage at 50 or 70°F (10 or 21°C). Values are means with standard 

deviations in parentheses. Absence of a superscript indicates no significant 

difference (P > 0.05) from initial value. 

Storage Treatment Log CFU per piece 

Conditions E. coli O157:H7 Salmonella spp. 

At inoculation 5.2 (0.1) A 5.0 (0.2) A 

Initial (24 h, 41°F) 4.4 (0.1) 4.3 (0.1) 

2 h at 50°F 4.8 (0.1) 4.6 (0.2) 

4 h at 50°F 4.8 (0.1) A 4.5 (0.2) 

6 h at 50°F 4.7 (0.1) 4.5 (0.1) 

1 h at 70°F 4.8 (0.1) 4.6 (0.1) 

3 h at 70°F 4.9 (0.2) A 4.7 (0.2) A 

5 h at 70°F 4.7 (0.1) 4.5 (0.1) 

A

Value is significantly different (P < 0.05) from initial value. 

67 
Report #4 Survival of Listeria monocytogenes on head cheese 

Validation of Head Cheese Formulation as an Antimicrobial Agent 

Against Listeria monocytogenes 

Steven C. Ingham1*, Jill A. Losinski1, and Dennis R. Buege2 

University of Wisconsin-Madison 

1

Department of Food Science 

2 Department of Animal Sciences 

* Corresponding author: 

1605 Linden Drive, Madison, WI 53706-1565 

Phone 608-265-4801, fax 608-262-6872, e-mail scingham@wisc.edu 

68 
INTRODUCTION 

The formulation of head cheese involves addition of large amounts of vinegar to 

pieces of meat. The finished product typically has a pH below 4.6 and, in some 

cases, the pH may be less than the 4.4 level which is cited as the minimum pH 

allowing growth of Listeria monocytogenes (2). At the request of a head cheese 

processor, the present study was conducted to validate a traditional head cheese 

manufacturing formulation as an effective antimicrobial agent against L. 

monocytogenes. Such a validation would allow the processor to operate under 

Alternative 2 of the USDA interim final rule addressing the control of Listeria 

monocytogenes on ready-to-eat meat and poultry products (1). 

MATERIALS AND METHODS 

Overview Head cheese from three different lots was inoculated with a 

multi-strain cocktail of Listeria monocytogenes. After samples were inoculated 

they were vacuum packed, and stored at 41°F (5°C). Three inoculated samples 

and one uninoculated sample were analyzed at day 0, day 8, and day 31 to 

determine the number of L. monocytogenes cells present. 

Head Cheese Three loaves of head cheese representing three separate 

lots were shipped overnight to the laboratory in cooler boxes with ice packs and 

stored at 41°F 5°C until used. The pH values of the three lots were 4.4, 4.2, and 

4.4. For this study, each loaf of head cheese was divided into 10 slices with 

69 
dimensions of 4.25 inches x 2.25 inches x 0.75 inches (10.8 cm x 5.7 cm x 1.9 

cm). 

Preparation of Inoculum Table 1 shows the strains used in this study. 

Frozen stock cultures were maintained in Brain Heart Infusion (BHIB; Difco, 

Becton Dickinson, Sparks, MD) with 10% (v/v) added glycerol (Fisher Scientific, 

Itasca, IL). Working cultures were prepared by growing each strain for two 

passages in BHIB and then streaking on Brain Heart Infusion Agar (BHIA; Difco). 

Following growth on BHIA for 24 h at 95°F (35°C), the working cultures were 

stored at 41°F (5°C). A colony of each strain was streaked on BHIA and grown 

for 24 h at 95°F (35°C), following which a colony of each culture was grown 

separately in 9 ml of BHIB for 24 h at 95°F (35°C). All cultures were combined, 

and then centrifuged at 8,000 x g for 10 minutes. Each resulting pellet was then 

re-suspended to original volume in Butterfield’s Phosphate Diluent (BPD, NelsonJameson, Marshfield, WI). 

Table 1. Organisms used in this study. 

Microorganism Strain Designation Original Source 

Listeria monocytogenes Scott Aa Human isolate- MA epidemic 

Listeria monocytogenes LM 101a

 Hard salami 

Listeria monocytogenes LM 108a

 Hard salami 

Listeria monocytogenes LM 310a

 Goat cheese 

Listeria monocytogenes V7a

 Raw milk a Obtained from the laboratory of Eric Johnson, Food Research Institute, University of WisconsinMadison 

70 
Inoculation of Head Cheese For inoculation, slices of head cheese 

were placed on aluminum foil that had previously been treated with 70% (v/v) 

ethanol in a laminar flow bio-safety hood. Each slice was inoculated with 0.01 oz 

(0.3 ml) of a 1:10 dilution (BPD) of the cocktail. The inoculum was spread evenly 

over the surface of the piece of head cheese using a sterile bent plastic rod 

(Daigger, Inc., Vernon Hills, IL). After inoculation, the pieces were allowed to dry 

for 15 min., flipped, and the procedure was repeated on the other side. Then, 

slices were aseptically transferred to a vacuum-packaging bag (FoodSaver, Tilia, 

Inc., San Francisco, CA), vacuum-packaged, and stored at 41°F (5°C). 

Uninoculated control slices of head cheese were packaged and stored in the 

same way. 

Enumeration of Inoculum Organisms After inoculation, enumeration for 

three inoculated samples was done. For enumeration, each vacuum sealed bag 

was first treated with 70% ethanol, then opened using scissors that had been 

flame sterilized. Next, a 1 inch x 1 inch x 0.25 inch section was aseptically 

excised from one randomly chosen side of the slice and placed into a whirl pack 

bag. Then, 3.3 oz (99 ml) of BPD was added to the sample bag and the contents 

were stomached for 2 minutes on medium speed using a Stomacher 400 lab 

blender (Fisher Scientific, Itasca, IL). Subsequent dilutions were made in BPD 

and spread-plated on LSA agar (Oxoid, Inc., Ogdensburg, NY) with added 

Listeria Selective Supplements (Oxford formulation; Oxoid) for enumeration of L. 

monocytogenes. LSA plates were incubated at 95°F (35°C) for 48 h, typical 

71 
colonies (brown to black colonies with a zone of black precipitate) were counted, 

and the logarithm of Colony-Forming Units (log CFU) was calculated for each 

sample. In addition to the enumeration described above, day 8 samples were 

also diluted and plated on BHIA, incubated 48 h at 95°F (35°C). This analysis 

provided an estimate of all aerobic bacteria on the product surface, including 

injured (but viable) and uninjured L. monocytogenes. To confirm that colonies 

counted on LSA were L. monocytogenes , one typical colony per plating per lot of 

head cheese was transferred to BHIA and incubated for 24 h at 95°F (35°C). 

Presumptive L. monocytogenes colonies from each BHIA plate were tested for 

Gram reaction, cell morphology, oxidase reaction, and biochemical 

characteristics (API Listeria; bioMerieux, Inc., Hazelwood, MO). Throughout the 

study, all presumptive colonies were confirmed as the L. monocytogenes. 

RESULTS AND DISCUSSION 

The results (Table 2) clearly show that L. monocytogenes rapidly died off on the 

surface of the head cheese. Population decreases after 8 days averaged about 

2.8 logs as determined using LSA and 2.5 logs as determined with BHIA plating. 

All colonies observed on BHIA after the day 8 plating had typical L. 

monocytogenes morphology. Counts on BHIA after 8 days of refrigeration were 

0– 0.4 logs higher than on LSA, indicating the presence of some injured, but 

viable, L. monocytogenes cells on the head cheese. However, by 31 days no 

survivors were detected on LSA. In order to claim that an antimicrobial agent is 

effective in preventing growth of L. monocytogenes on a ready-to-eat meat or 

72 
poultry product, the agent must not allow more than a 1 log increase in L. 

monocytogenes populations (2). In the head cheese, the L. monocytogenes 

population decreased, so the processor may consider the product formulation to 

be an effective antimicrobial agent. Other head cheese processors should 

determine the typical pH of their product. If the pH is within the 4.2 – 4.4 range 

described in the present study, the processor may consider the product 

formulation to be an effective antimicrobial agent, allowing operation under 

Alternative 2 of the USDA regulations (1). 

Table 2. Log CFU/g (standard deviation in parentheses) of Listeria 

monocytogenes. 

A B C 

Day 0 5.9 (0.1) 5.7 (0.1) 5.8 (0.1) 

Day 8 3.2 (0.3) 2.0a (0) 3.1 (0.4) 

Day 8 

(BHIA) 

3.6 (0.3) 2.2 (0.4) 3.1 (0.1) 

Day 31 0.9b (0.0) 0.9b (0.0) 0.9b (0.0) 

a- no LM detected on 1:100 dilution, assigned value of 1.9 log CFU/g 

b- no LM detected on 1:10 dilution, assigned value of 0.9 log CFU/g 

ACKNOWLEDGEMENTS This study was funded by the United States 

Department of Agriculture, Food Safety and Inspection Service, contract USDAFSIS-27-2003. 

73 
REFERENCES 

1. 1. United States Department of Agriculture, Food Safety & Inspection 

Service. 2003. Control of Listeria monocytogenes in ready-to-eat meat 

and poultry products; final rule. Federal Register: June 6, 2003 (Vol. 68, 

No. 109, pp. 34207-34254). 

2. United States Department of Agriculture, Food Safety & Inspection 

Service. Compliance guidelines to control Listeria monocytogenes in 

post-lethality exposed ready-to-eat meat and poultry products. 

http://www.fsis.usda.gov/OPPDE/rdad?FRPubs/97-

013F/CompGuidelines.doc. Accessed 19 April 2004. 

74 
Report #5. Survival of Listeria monocytogenes on Ready-to-Eat Meat 

Products 

Modified version of article accepted for publication in the Journal of Food 

Protection 

Survival of Listeria monocytogenes During Storage of Ready-to-Eat Meat 

Products Processed by Drying, Fermentation, and/or Smoking 

Steven C. Ingham*1, Dennis R. Buege2, Brenda K. Dropp1, and Jill A. Losinski1 

University of Wisconsin-Madison 

1

 Department of Food Science 

2

 Department of Animal Sciences 

*Corresponding author 

University of Wisconsin-Madison, Department of Food Science 

1605 Linden Drive, Madison, WI 53706-1565 

Phone 608-265-4801 

Fax 608-262-6872 

e-mail scingham@wisc.edu 

75 
ABSTRACT 

The survival of Listeria monocytogenes was evaluated on 15 ready-to-eat 

meat products made using drying, fermentation, and/or smoking. The 

products were obtained from 6 processors and included summer sausage, 

smoked cured beef, beef jerky, snack stick, and pork rind/cracklin products. 

The water activity (aw) of the products ranged from 0.27 (pork rinds/cracklins) 

to 0.98 (smoked cured beef slices). Products were inoculated with a 5-strain 

cocktail of L. monocytogenes, re-packaged under either vacuum or air, and 

then stored at either room temperature (70°F / 21°C) or under refrigeration 

(41°F / 5°C) for 4- 11 weeks. Numbers of L. monocytogenes fell for all 

products during storage, ranging from a decrease of 0.8 log CFU on smoked 

cured beef slices during 11 weeks under vacuum at 41°F (5°C) to a decrease 

of 3.3 log CFU on a pork rind product stored 5 weeks under air at 70°F 

(21°C). All of the products tested could be produced under Alternative 2 of 

the United States Department of Agriculture (USDA) regulations mandating 

control of L. monocytogenes on ready-to-eat meat and poultry products. For 

many of the products, 1 week of post-processing storage prior to shipment 

would act as an effective post-lethality treatment and would allow processors 

to operate under Alternative 1 of these regulations. 

76 
INTRODUCTION On June 6, 2003, the United States Department of 

Agriculture (USDA) published an interim final rule addressing the control of 

Listeria monocytogenes on ready-to-eat (RTE) meat and poultry products (2). 

This rule went into effect October 6, 2003 and has already had a major effect on 

processors of these products. The rule is intended to encourage processors of 

RTE products to take one or more specific steps to ensure the absence of L. 

monocytogenes from their products. These steps range from focused sanitation 

procedures to adding formulation or processing steps designed to kill L. 

monocytogenes or inhibit its growth. The processor is also required to perform 

testing for L. monocytogenes or Listeria spp. on food contact surfaces in the 

area of the plant in which RTE products are handled after cooking. The amount 

of testing is related to the types of RTE products made, product ingredients, and 

how the products are processed and handled. In particular, the rule requires 

processors of RTE meat and poultry products to adopt one of three designated 

“Alternatives” to control L. monocytogenes on their products. The Alternatives 

involve varying levels of control and microbiological testing. In Alternative 1, the 

processor uses a post-lethality treatment that reduces or eliminates L. 

monocytogenes AND an antimicrobial agent or process that suppresses or limits 

L. monocytogenes growth throughout product shelf-life. In Alternative 2, the 

processor uses either a post-lethality treatment that reduces or eliminates L. 

monocytogenes OR an antimicrobial agent or process that suppresses or limits 

L. monocytogenes growth throughout product shelf-life. Under Alternative 3, only 

sanitation measures are relied upon to control L. monocytogenes. 

77 
Most small-scale meat processors in Wisconsin have chosen Alternative 3 

for controlling L. monocytogenes. This alternative provides the least assurance of 

safety and requires the most frequent testing of food contact surfaces among the 

three alternatives. For many RTE meat products though, the reduction of water 

activity, accomplished through the addition of salt and cooking or drying, could 

serve as an antimicrobial process by making the finished product unsuitable for 

L. monocytogenes growth. Similarly, the reduction of pH via fermentation or 

addition of an acidulant, or surface-deposition of inhibitory compounds via 

smoking could also be effective antimicrobial processes. Items produced in 

these ways could thus fall under Alternative 2. Compliance guidance from USDA 

(3) has stated that an effective antimicrobial process will allow no more than a 

1.0 log increase in L. monocytogenes on an RTE product throughout its shelf-life. 

This guidance also summarized scientific studies indicating that L. 

monocytogenes will not multiply at a water activity of < 0.92 or a pH of <4.39. 

It is also possible that some RTE products could fall under Alternative 1, 

when the above-mentioned antimicrobial processing techniques are combined 

with short-term storage prior to distribution that effectively serves as a postlethality treatment. The compliance guidance from USDA states that an effective 

post-lethality treatment must reduce numbers of L. monocytogenes by at least 1 

log. 

The regulation requires that post-lethality treatments must be scientifically 

validated and that evidence must be provided to substantiate the effectiveness of 

78 
antimicrobial processes. Unfortunately, most small-scale processors are unable 

to provide this evidence. 

The objective of the present study was to evaluate the survival, and 

perhaps death, of inoculated L. monocytogenes during storage (after repackaging) on a variety of RTE meat products made using drying, fermentation 

and/or smoking techniques. Information obtained in the study could thereby 

provide evidence needed by processors to implement Alternatives 1 or 2 in their 

RTE meat product operations. 

MATERIALS AND METHODS Packages of RTE meat products were 

submitted by six processors. Product ingredients are summarized in Table 1. 

Depending on the product, 1 – 3 lots of product were tested. A representative 

sample from each lot of product was vacuum packaged and sent to a commercial 

testing laboratory to be analyzed for water activity, pH, and % water-phase salt 

(forced air oven determination of moisture – AOAC method 950.46Bb and 

potentiometric method for salt – AOAC method 980.25; 1). For each product, the 

lowest % water-phase salt value, and the highest water activity and pH values, 

i.e. conditions least restrictive to microbial growth, are reported in Table 2. 

Because post-lethality contamination of RTE products by L. monocytogenes will 

occur only on the product surface, 0.6 cm thick sections of product, with surface 

dimensions of 1.5 in x 1.5 in (3.7 x 3.7 cm), were cut from each product using a 

knife [previously sanitized using 70% (v/v) ethanol] and placed in a biosafety 

hood on aluminum foil that had previously been sanitized with ethanol and 

79 
ultraviolet light. For beef jerky, the 1.5 in x 1.5 in (3.7 x 3.7 cm) pieces were cut 

from individual jerky slices. For the pork rind/cracklin products, existing individual 

pieces, each about 1 in x 1 in (2.5 x 2.5 cm), were individually inoculated. 

The L. monocytogenes strains used in this study were obtained from the 

laboratory of Dr. Eric Johnson at the Food Research Institute, University of 

Wisconsin-Madison, and are listed in Table 3 . Stock cultures were maintained 

at -4°F (-20°C) in Brain Heart Infusion broth (BHIB; Difco, Becton-Dickinson, 

Sparks, MD) with 10% (w/v) added glycerol (Fisher Scientific, Itasca, IL). 

Working cultures, maintained at 40°F (4°C) on Brain Heart Infusion agar (BHIA; 

Difco) were prepared monthly from frozen stock cultures. To obtain a working 

culture, a strain was cultured twice successively at 95°F (35°C) for 18-24 h in 

BHIB, streaked to a BHIA plate, incubated at 95°F (35°C) for 18-24 h and 

examined for purity, and then stored at 40°F (4°C). Inoculation cultures were 

prepared for each strain by transferring a loopful of growth from the working 

culture plate to 0.3 oz (9 ml) of BHIB and incubating at 95°F (35°C) for 20-24 h. 

To prepare the 5-strain inoculum cocktail, the BHIB cultures were combined and 

distributed evenly into two 1.7-oz (50-ml) sterile plastic centrifuge tubes, and 

centrifuged for 10 minutes at 5,000 x g. The supernatant in both tubes was 

decanted and the pellets were resuspended to the original volume in Butterfield’s 

phosphate diluent (BPD; Nelson Jameson, Marshfield, WI). The resulting 

cocktail was serially diluted in BPD and plated to determine cell concentration. 

To inoculate the meat or pork rind/cracklin pieces, a 0.0008 oz (0.025 ml) volume 

of the undiluted cocktail was pipetted onto the product surface and distributed as 

80 
evenly as possible using a sterile plastic spreader. The product pieces were then 

allowed to dry for at least 15 minutes and then vacuum packaged (0.8 atm.; Food 

Saver bags and packaging machine; Tilia, Inc.; San Francisco, CA) and stored at 

either 41 or 70°F (5 or 21°C). Pork rind/cracklin pieces were allowed to dry and 

then stored aerobically in zip-lock plastic bags at 70°F (21°C). 

At the start of the study and after 1and 4, 5, or 11 weeks of storage, samples 

were analyzed for the number of L. monocytogenes cells per sample. The 

sample bag was aseptically opened, BPD (1.7 oz / 99 ml) was added, and the 

sample was stomached for 2 minutes at medium speed (Stomacher 400 lab 

blender; Fisher). Serial dilutions were made in BPD as needed. For the initial 

dilution, 0.03 oz (1.0 ml) was distributed for spread-plating among three plates 

(0.3, 0.3, and 0.4 ml) of Listeria Selective Agar (LSA; Oxoid, Ogdensburg, NY) 

with Listeria Selective Supplements (Oxford formulation; Oxoid). From the 

original dilution and each subsequent dilution, 0.003 oz (0.1 ml) was spread on 

one LSA plate per dilution. Plates were incubated at 95°F (35°C) for 48 h and 

then examined for typical L. monocytogenes colonies (small-medium, greybrown-to-black; raised, flat, or sunken colonies surrounded by a black precipitate 

zone). Replica plating from Nutrient agar (Difco) to LSA was performed for some 

summer sausage samples but there was little evidence of cell injury. 

Furthermore, injured cells would be unlikely to survive the low-pH stress of 

human gastric juice if products were ingested. Therefore, direct plating on LSA 

was used throughout the study. For each product lot analyzed, one presumptive 

L. monocytogenes colony was selected at each sampling time for confirmation 

81 
testing. The colony was transferred to Nutrient Agar and after incubation was 

tested for Gram stain reaction, cellular morphology, oxidase activity, and 

biochemical characteristics (API Listeria kit, bioMerieux, Hazelwood, MO). 

Throughout the study, all presumptive isolates were confirmed as L. 

monocytogenes. 

With the exception of the pork rind/cracklin products, where only a small 

amount of sample was available, three pieces were analyzed at each sampling 

time for each lot of a particular product. For the pork rind/cracklin products, only 

one sample was analyzed per sampling time for each lot. The log CFU was 

calculated for each piece. Then the mean log CFU per piece was calculated for 

each lot and the mean of all lots tested was determined for the product at that 

sampling time. A value of 0.9 log CFU/g was assigned when no colonies were 

present for the least dilute plating. 

RESULTS AND DISCUSSION The RTE meat products varied widely in water 

activity, pH and % water-phase salt (Table 2). Given that higher water activity 

and pH and lower % water-phase salt would increase the likelihood of L. 

monocytogenes growth, the product judged most likely to allow growth of this 

pathogen was the smoked cured beef slices and the products judged least likely 

to support growth were the pork rind/cracklin products and beef jerky. Numbers 

of L. monocytogenes recovered from stored products were consistent with these 

compositional factors. Levels of L. monocytogenes fell rapidly on the pork 

rind/cracklin products during room temperature storage (Table 4), with decreases 

82 
of 1.2 – 2.1 log CFU in the first week of storage, and subsequent decreases to 

levels at or near the detection limit for samples stored 5 weeks. Similar results 

were obtained for the beef jerky (Table 4). Processing RTE products to yield 

water activity of < 0.75, combined with 1 week of 21°C storage, appears to 

effectively allow the processor to operate under Alternative 1, with the processing 

technique as the antimicrobial process and the one-week storage as the postlethality treatment. It is not known whether the decrease in L. monocytogenes 

numbers during storage occurred at a uniform rate or was early in the one-week 

period. The latter situation would result in a shorter pre-shipment storage period 

serving as the post-lethality treatment. 

The sausage products (summer, elk, buffalo) had maximum water activity of 

0.94 – 0.96, considerably higher than for beef jerky. However, fermentation of 

the summer sausage products during processing resulted in a lower finished 

product pH than for the pork rind/cracklin products and jerky. (Table 2). Neither 

the water activity nor the pH of these products was low enough to predict, based 

on USDA compliance guidance, that L. monocytogenes would not grow. The 

combination of somewhat reduced water activity and pH seemed to effectively 

inhibit growth and cause death of L. monocytogenes. L. monocytogenes 

decreased in numbers by >1.0 log at room temperature (Table 4) or during 

refrigeration (Table 5). These results strongly suggest that sausage products, 

with a mandatory one-week pre-distribution storage period, could also be 

produced under Alternative 1. To do so, processors would be required to either 

ensure that their summer sausage had water activity and pH at least as low as 

83 
those products used in the present study, or have a challenge study conducted to 

validate the post-lethality treatment for their products. 

Somewhat less death of L. monocytogenes occurred during storage of snack 

stick products (Table 5). It is clear that the snack stick products could be 

produced under Alternative 2; case-by-case studies would be necessary to 

determine if Alternative 1 could be chosen for these products. Roomtemperature storage of snack stick products was not evaluated, although many 

products of this type are stored at ambient temperatures. 

The intact and sliced smoked cured beef had the highest water activity and 

lowest % water-phase salt of the products studied (Table 2). As expected, L. 

monocytogenes survival was best on the sliced smoked cured beef product 

(Table 5) with decreases of only 0.8 log CFU during 11 weeks of refrigerated 

storage. Surprisingly, L. monocytogenes numbers decreased 3.0 log CFU on the 

surface of intact smoked beef during this same storage period. This difference in 

survival could reflect the somewhat lower water activity of the intact product, 

localized areas on the product surface possibly having even lower water activity, 

and perhaps the presence of anti-microbial smoke-derived compounds on the 

surface of the smoked beef product. The smoked cured beef results suggest that 

processing of these products should be done under Alternative 2 of the 

regulations. 

 In conclusion, the smoking, cooking, and drying processes used to make the 

tested products can be considered effective antimicrobial processes under the 

USDA regulations mandating control of L. monocytogenes on ready-to-eat meat 

84 
and poultry products. To verify that their own processing techniques allow 

operation under Alternative 2, processors should either verify that their products 

have water activity, pH, % water-phase salt levels at least as restrictive as those 

in the present study, or have a challenge study conducted to validate the lack of 

L. monocytogenes growth on their products. For many of the products studied, a 

one- week period of post-packaging room temperature storage prior to shipment 

could act as an effective post-lethality treatment and would allow processors to 

operate under Alternative 1 of the USDA L. monocytogenes regulations. Again, 

processors should verify similar product composition or conduct a challenge 

study to validate the post-lethality treatment lethality. 

ACKNOWLEDGMENTS The authors acknowledge the cooperation of 

processors providing product. This work was partially supported by the United 

States Department of Agriculture, Food Safety and Inspection Service, contracts 

USDA-FSIS-C-27-2003 and USDA-FSIS-C-37-2003. 

REFERENCES 

1. AOAC International. 1998. pp. 39.1.02, 46.5.02. Official methods of 

analysis of AOAC International, 16th edition, 4th revision, Volume II. p. 

Cunniff (ed.). AOAC International, Gaithersburg, MD. 

85 
2. United States Department of Agriculture, Food Safety & Inspection 

Service. 2003. Control of Listeria monocytogenes in ready-to-eat meat 

and poultry products; final rule. Federal Register: June 6, 2003 (Vol. 68, 

No. 109, pp. 34207-34254). 

3. United States Department of Agriculture, Food Safety & Inspection 

Service. Compliance guidelines to control Listeria monocytogenes in 

post-lethality exposed ready-to-eat meat and poultry products. 

http://www.fsis.usda.gov/OPPDE/rdad?FRPubs/97-

013F/CompGuidelines.doc. Accessed 19 April 2004. 

86 
Table 1. Ingredients of ready-to-eat meat products evaluated for survival of 

inoculated Listeria monocytogenes during storage (after re-packaging). 

Product Processor Ingredient Summary 

Description 

Smoked cured A Beef, cured with water, salt, sugar, sodium 

beef erythorbate, sodium nitrite 

piece/slices 

Summer A Beef and pork, salt, dextrose, natural spices, lactic 

sausage acid starter culture, sodium erythorbate, sodium 

nitrite 

Summer B Beef, beef hearts1

, salt, corn syrup solids, dextrose, 

sausage spices, mustard seed, sodium erythorbate, lactic 

acid starter culture, sodium nitrite 

Summer C Beef, pork, water, salt, corn syrup solids, dextrose, 

sausage spices, ground mustard, lactic acid starter culture, 

sodium erythorbate, sodium nitrite 

Buffalo C Buffalo, pork, water, salt, sugar, pepper, mustard 

summer seed, garlic, monosodium glutamate, sodium 

sausage erythorbate, sodium nitrite 

Elk summer C Elk, pork, water, salt, sugar, black pepper, mustard 

sausage seed, garlic, monosodium glutamate, lactic acid 

starter culture, sodium erythorbate, sodium nitrite 

87 
Venison F Venison, pork, water, salt, sugar, corn syrup solids, 

snack sticks spices, dextrose, dehydrated garlic, red pepper, 

lactic acid starter culture, sodium nitrite 

Beef snack A Beef, salt, dextrose, natural spices, natural 

sticks flavoring, lactic acid starter culture, sodium 

erythorbate, sodium nitrite 

Pork rinds D Pork rinds, seasoning2 

Pork cracklins D Pork cracklins (fried out pork fat with attached skin), 

salt 

Beef jerky E Beef, water, salt, flavoring, sodium nitrate and 

sodium erythorbate 

1 One other lot also contained pork; another tested lot also contained pork and 

garlic. 

2 Seasonings differed for regular, smoke flavor, and BBQ flavor. 

88 
Table 2. Chemical and physical characteristics of ready-to-eat meat products 

evaluated for survival of inoculated Listeria monocytogenes during storage (after 

re-packaging). 

Product Processor aw pH water-phase 

Description salt (%)a 

Smoked cured beef piece A 0.96 5.5 2.9 

Smoked cured beef slices A 0.98 5.6 2.5 

Summer sausage A 0.96 4.7 3.9 

Summer sausage B 0.95 4.9 5.2 

Summer sausage C 0.96 4.8 5.0 

Buffalo sausage C 0.95 5.2 6.5 

Elk sausage C 0.96 5.3 4.5 

Venison snack sticks F 0.91 4.8 7.6 

Beef snack sticks, small A 0.95 5.0 5.6 

Beef snack sticks, large A 0.93 5.0 5.9 

Pork rinds D 0.29 6.0 56.9 

Pork rinds, smoke flavor D 0.27 6.1 60.7 

Pork rinds, BBQ flavor D 0.27 6.1 69.3 

Pork cracklins D 0.28 6.7 69.2 

Beef jerky E 0.75 5.6 14.4 

a

 Also referred to as “brine content”. 

89 
Table 3. Strains of Listeria monocytogenes used to inoculate ready-to-eat 

meat products prior to re-packaging and storage. 

Genus and species Strain Designation Original Source 

Listeria monocytogenes Scott A Human outbreak isolate 

Listeria monocytogenes LM 101 Hard salami 

Listeria monocytogenes LM 108 Hard salami 

Listeria monocytogenes LM 310 Goat cheese 

Listeria monocytogenes V7 Raw milk 

90 
Table 4. Survival of inoculated L. monocytogenes on ready-to-eat meat 

products stored after re-packaging under air or vacuum at 70°F (21°C). 

Product Log CFU/sample (mean) 

Description with n and range in parentheses 

(processor) Start 1 week 5 weeks 

Summer sausage (B) 4.2 (1,0) 1.0 (1,0) 0.9 (1,0)ab 

Summer sausage (C) 3.4 (1,0) 1.5 (1,0) 0.9 (1, 0) 

Elk sausage (C) 4.0 (1,0) 2.4 (1,0) 0.9 (1,0) 

Buffalo sausage (C) 3.1 (1,0) 0.9 (1,0) 0.9 (1,0) 

Pork rinds (D) 4.3 (2, 0.1) 2.2 (2, 0.6) 1.1 (2,0.4) 

Pork rinds, smoke flavor (D) 4.4 (1, 0) 2.7 (1, 0) 1.7 (1,0) 

Pork rinds, BBQ flavor (D) 4.2 (1,0) 3.0 (1,0) 0.9 (1,0) 

Pork cracklins (D) 4.2 (2,0.4) 2.6 (2,1.6) 1.1 (2, 0.4) 

Beef jerky (E) 3.6 (3, 1.2) 1.2 (3, 0.2) 0.9 (3,0) 

a 0.9 is value assigned when no cells were detected. 

b

Value obtained after 4 weeks of 70°F (21°C) storage. 

91 
92 
Table 5. Survival of inoculated L. monocytogenes on ready-to-eat meat products stored after re-pa

vacuum at 41°F (5°C). Product Log CFU/sample (mean) Description with number of lots and range in parentheses 

(processor) Start 1 week 4 weeks 5 weeks 11 weeks 

Smoked beef piece (A) 4.5 (2, 0.2) 3.7 (2, 0.9) NTa NT 1.5 (2,1.1) 

Smoked beef slices (A) 4.3 (2, 0.1) 4.2 (2, 0.2) NT NT 3.5 (2,0.5) Summer sausage (A) 3.6 (3, 0.6) 2.3 (3, 0.4) 0.9 (3, 0) NT NT 

Summer sausage (B) 3.9 (3, 0.3) 1.5 (3, 1.8) NT NT 0.9 (3, 0) Summer sausage (C) 3.4 (1,0) 1.5 (1,0) NT NT 0.9 (1,0) 

Buffalo sausage (C) 3.7 (1,0) 2.6 (1,0) NT NT 1.5 (1,0) 

Elk sausage (C) 3.8 (1,0) 3.2 (1,0) NT NT 2.4 (1,0) Venison snack sticks (F) 4.0 (1, 0) 3.6 (1, 0) NT 1.1 (1, 0) NT 

Beef snack sticks, small (A) 3.5 (2, 0.2) 2.5 (2, 0.3) NT 2.1 (2, 1.0) NT 

Beef snack sticks, large (A) 3.7 (1, 0) 3.0 (1,0) NT 2.0 (1, 0) NT 
aNot tested.