Comparison of Conventional Plating Methods and

1084 Journal of Food Protection, Vol. 60, No.9, 1997, Pages 1084-1088 Copyright

©, International

Association

of Milk, Food and Environmental

Sanitarians

Comparison of Conventional Plating Methods and Petrifilm for the Recovery of Microorganisms in a Ground Beef Processing Facilityt R. H. LINTON, * W. G. EISEL, and P. M. MURIANA Department of Food Science, 1160 Smith Hall, Purdue University, West Lafayette, IN 47907, USA (MS# 96-240: Received 10 September

ABSTRACT The objective of this study was to compare recovery of microorganisms for various beef samples and beef contact surfaces using conventional pour plating techniques and Petri film methods. Comparisons for aerobic plate count (APC), coliform count (CC), and Escherichia coli count (ECC) were done for 104 fresh or frozen retail cuts and 56 food surface or food contact surfaces. Samples were taken at a midwestern retail ground beef processing plant during a 12-month project. APC comparisons were made for pour plating using Trypticase soy agar versus Aerobic Plate Count Petrifilm. CC and ECC were compared for pour plating using violet red bile + MUG agar versus E. coli Petrifilm. Overall, paired t tests revealed a significantly higher recovery for APC from fresh and frozen beef samples using the pour plating technique (P :S 0.05). No significant differences (P > 0.05) were observed for CC from fresh and frozen meat samples. Recovery of E. coli from many beef samples was better using Petrifilm. Significantly higher ECCs were observed from fresh and frozen meat samples using Petrifilm compared to the pour plating technique (P:S 0.05). For food surfaces and food contact surfaces, a comparison between pour plating and Petrifilm was done for aerobic plate count. No significant differences (P > 0.05) in recovery could be found between methods. A comparison between neutralizing buffer and letheen broth for recovery of surface microorganisms was done for both the APC pour plating method and APC Petrifilm. In both cases, recovery when using letheen broth was significantly (P :S 0.05) higher than neutralizing buffer. Because it is convenient and gave comparative results, Petrifilm offers a good alternative for environmental microbial testing and red meat product testing. Key words: Pour-plate, Petrifilm, coliform, Escherichia coli

In recent years, there has been a growing concern regarding the safety and quality of meat and poultry products. The most recognized approach for controlling food safety and quality is to establish hazard analysis critical control point (HACCP) programs and good manufacturing

* Author

for correspondence.

Tel: 765-494-6481;

Fax: 765-494-7953. Research

t Journal paper no. 15171 of Purdue University, Agricultural Programs.

1996/Accepted

2 December

1996)

practices (GMPs), respectively. HACCP and GMP programs have been used for over 20 years in food processing plants. More recently, HACCP programs have been broadened to cover the entire food production chain-from the farm to the plate of consumers. Increased emphasis for HACCP program development is being directed toward farm, slaughter, and harvest operations. An integral component of HACCP programs is periodic testing of microbial contamination levels. Common sampling protocols include microbial assessment from products at various steps in the food process, from food contact surfaces, from water, and from air. Microbial sampling plans differ greatly depending on the food product and the food process. Current legislation has been published by the United States Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) which includes several changes to the meat and poultry inspection program (2). Under the new legislation, meat and poultry plants will be required to adopt and carry out HACCP programs. HACCP programs will be continually monitored by USDA inspectors. Mandatory testing for E. coli will also be required six months after the final rule for establishments that slaughter meat and poultry. The required frequency of E. coli testing will be based on production volume. E. coli testing for poultry products will be done using a carcass rinse, whereas carcasses of beef and pork will be sampled using a surface sponge technique. The rationale for testing for E. coli is that the E. coli results may provide useful information regarding plant sanitation, potential presence of other enteric bacteria, and process control. Sanitation standard operating procedures (SSOPs) will also be required as part of the regulations. To establish these new programs, a microbial sampling plan for meat and poultry slaughter and processing plants would likely include periodic monitoring of aerobic mesophilic bacteria, coliform bacteria, and E. coli. The number of aerobic bacteria in beef samples would provide relative information regarding the expected product shelf life. The number of aerobic bacteria recovered from food contact surfaces can be used to assess the cleanliness of the surface and hence the probability of cross-contamination from the food contact surface to a food. Coliform and E. coli counts

COMPARISON

OF CONVENTIONAL

from beef products and food contact surfaces can be used to indicate potential safety and quality. High coliform and E. coli counts suggest that other foodborne pathogens of fecal origin may be present. High coliform and E. coli counts on food contact surfaces can be used to indicate insufficient cleaning and sanitizing programs (7). Traditional methods for the enumeration of aerobic bacteria, coliforms, and E. coli include sampling a food or food contact surface, followed by pour plating on a suitable microbial medium (8). For in-house microbial analysis of foods, this type of analysis may be time-consuming (i.e., requiring media preparation) and more difficult to perform. An alternative method is the use of Petrifilm (3M Health Care, St. Paul, MN). Petrifilm is a ready-made dehydrated microbial medium contained on a paper surface covered by a plastic film. The use of Petrifilm to recover microorganisms in a food plant may be more practical compared to pour plating techniques. When using Petrifilrn, no media preparation is required, incubator and storage space is reduced, and enumeration of colonies is facilitated because Petrifilm contains counting grids and a visible indicator dye. Comparisons of pour plating and Petrifilm have been done for various products. Ingham and Moody (3) compared "Standard Methods" pour plates to Petrifilm "Standard Methods" films and compared a five-tube E. coli most probable number (MPN) procedure to Petrifilm E. coli count films. No significant differences in aerobic bacterial counts (P > 0.05) were found between pour plating and Petrifilm. However, Petri film appeared to be a more sensitive method, compared to MPN, for detection of E. coli. Chain and Fung (1) compared Redigel (3M Health Care, St. Paul, MN), Petrifilm, spiral plating, and pour plating using standard plate count agar for several different foods (chicken, ground beef, ground pork, shelled pecan, raw milk, thyme, and flour). The results showed that all five methods were highly comparable (r = 0.97). Smith et al. (6) compared Petrifilm and pour plating for the enumeration of aerobic mesophilic bacteria in retail ground beef samples. Total colony-forming units were determined for 119 retail ground beef samples during a nine-week study. The mean counts using the pour plate method and Petrifilm method were 6.11 log CFU/g and 5.85 log CFU/g, respectively. Mean counts using the pour plate method were 0.25 log higher than those with Petrifilm (P ~ 0.01). It is evident that more microbial sampling programs will be established in the near future for meat slaughter and meat processing plants. Whether samples are done "inhouse" or sent out to a testing laboratory, microbial samples for meat products and meat contact surfaces will require additional time and money. Any measures that can be taken to reduce time and money, without disrupting the integrity of HACCP programs, will be useful to meat slaughter and processing operations. The objective of this study was to evaluate conventional pour plating techniques versus Petrifilm methods for recovery of aerobic bacteria, coli forms, and E. coli from various fresh or frozen beef products and for food contact surfaces in a beef processing facility.

PLATING AND PETRIFILM

1085

MATERIALS AND METHODS Description of the processing plant A 12-month study was done at a midwestern ground beef processing facility. Raw material was received as carcass beef and boxed beef (chuck, round, or sirloin). Carcass beef was then trimmed, cut into retail cuts, packaged, and stored at refrigeration (O°C). The trim was then combined with boxed beef and ground to fabricate ground beef of a desired fat level. Ground beef was packaged in 5-lb (2.26-kg) chubs and then held at refrigeration (O°C). Fresh andfrozen product sampling procedure Product sampling included a microbial assessment of fresh and frozen beef samples. The term "fresh" is used to describe samples that were taken at the plant during the processing of ground beef. Fresh products were collected every two weeks. Fresh product samples were taken along the processing line from incoming product to finished product to represent all areas of the process. For each sample, a sample weighing more than 25 g was aseptically taken (with a sterile spoon, knife, etc.), placed into a sterile sampling bag, and immediately placed into a mobile incubator held at 4°C. "Frozen" samples included various retail beef cuts, boxed beef, and ground beef samples. These samples were collected by personnel working in the plant, placed into sterile bags, and held at -20°C until analysis. Upon return to the laboratory, fresh product samples were weighed and aseptically trimmed to a sample weight of approximately 25 g. Each ofthe 25-g samples was then placed into a sterile stomacher bag, and 225 ml of 0.1 % (wt/vol) peptone buffer was added to make a 10-1 dilution. Samples were further diluted in 0.1 % peptone as needed, and plated in duplicate onto Trypticase soy agar (TSA) and violet red bile agar (VRBA) + methylumbelliferyl-l3-glucuronide (MUG) using a pour plate with overlay technique and onto Aerobic (APC) Petrifilm and E. coli Petri film. All media were incubated at 37°C. Coliforms and E. coli were enumerated from VRBA + MUG plates after a 24-h incubation period. Pink or purple colonies were recorded as coliforms, and those colonies that fluoresced under ultraviolet light were reported as presumptive E. coli. On E. coli Petrifilm, after 24 h, coliforms appeared pink, purple, or blue with gas production. The blue colonies with gas production were recorded as E. coli. For APCs, mesophilic aerobic bacteria were enumerated after 48 h from TSA and from APC Petrifilm. Frozen product samples were tested in the same manner as fresh products except that frozen samples were allowed to thaw overnight in a 4°C incubator prior to microbial analysis. In all cases, plate counts were determined and converted to log CFU/g (8). Table 1 lists beef product samples that were tested. Food contact surface and product surface sampling procedure For food surfaces and food contact surfaces, a surface swab technique was used as has been described (8). A sterile polypropylene template was used to sample a lO-cm2 surface area. A sterile calcium alginate swab was introduced into 10 ml of sterile neutralizing buffer (Difco Laboratories, Detroit, MI) or sterile letheen buffer (Difco), a lO-cm2 area was sampled, and the swab placed aseptically back into the respective buffer. Immediately after taking the sample, the tube containing the swab was then placed in a mobile incubator and maintained at 4°C until arrival at the laboratory. The elapsed time from collection of samples to plating was between 1 and 2 h after the samples were placed in the mobile incubator. Samples collected with neutralizing buffer or letheen broth were used for the pour plate technique or Petrifilm plating, respectively.

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LINTON, EISEL, AND MURIANA

TABLE 1. Fresh and frozen beef samples collected at the ground beefprocessing plant Sampling type

Description of sample

Fresh carcass beef Fresh carcass beef Fresh carcass beef Fresh carcass beef Fresh boxed beef Fresh boxed beef Fresh ground beef Fresh ground beef Fresh ground beef Fresh retail beef cuts Frozen carcass beef Frozen boxed beef Frozen boxed beef Frozen ground beef Frozen ground beef

Brisket area Flank area Round area Skirt area Cow Bull Course grind Fine grind Finished, packaged product Sirloin steak, rib eye steak, T-bone steak Cow Cow Bull Course grind Fine grind

Once at the laboratory, each tube was agitated on a Vortex mixer for 10 s to assure mixture of the sample. Samples were serially diluted as needed in 0.1 % (wt/vol) peptone buffer and plated in duplicate onto TSA and VRBA + MUG using a pour plate technique and onto APC Petri film and E. coli Petri film. All media were incubated at 37°C and enumerated as described above. Table 2 lists surface samples that were tested. Neutralizing buffer versus letheen broth for surface samples Neutralizing buffer is part of the recommended procedure when performing a surface swab followed by a pour plate TABLE 2. Surface samples collected at the ground beefprocessing plant Sampling location

Description of sample

Floor Floor Floor Floor Floor Floor Wall Wall Wall Wall Wall Wall Equipment, ground beef line Equipment, ground beef line Equipment, ground beef line Equipment, ground beef line Fabrication (cuts of beef) line Fabrication (cuts of beef) line Fabrication (cuts of beef) line Fabrication (cuts of beef) line Retail packaging line Retail packaging line Carcass beef Carcass beef Carcass beef Carcass beef Boxed beef

Loading dock Carcass cooler Trim cooler Ground beef line Fabrication (cuts of beef) line Retail packaging line Receiving truck Carcass cooler Trim cooler Ground beef line Fabrication (cuts of beef) line Retail packaging line Conveyor belt Stainless steel funnel Stainless steel bins Stainless steel under conveyer belt Conveyor belt Stainless steel plates Cutting boards Cutting knives Stainless steel plates Cutting board Brisket area Flank area Round area Skirt area Chuck

method (8). Letheen broth has been recommended for surface swab samples when followed by plating onto Petrifilm (5). The effects of these two buffers on recovery of bacteria from surfaces were studied for various food surfaces and food contact surface samples. For each surface sample, neutralizing buffer and letheen broth were used independently on adjacent food contact surfaces or product surfaces. Plating in each case was done for aerobic mesophilic bacteria only using the conventional pour plating techniques and Petrifilm. Surface samples for this comparison were the same as discussed above. Statistical analysis Comparisons of microbial counts for traditional pour plating versus Petrifilm plating for aerobic bacteria, coliform count, and E. coli were done separately for fresh and frozen product surfaces, and, for food contact surfaces and food surfaces. A comparison of microbial counts using letheen broth versus neutralizing buffer for aerobic bacterial counts was done for pour plating versus Petrifilm. In each situation, statistical differences at the 0.05 level of significance were evaluated using a pairwise comparison Student t test (IMP, SAS, Cary, NC).

RESULTS AND DISCUSSION The beef samples that were tested included 46 different fresh samples and 58 different frozen samples. Samples from both fresh and frozen beef were taken at all areas during production of ground beef from receipt of raw ingredients to completion of the finished product. Ranges and mean values for recoveries of mesophilic bacteria, coliform bacteria, and E. coli for the fresh and frozen samples are presented in Table 3. The average recovery values for aerobic mesophilic bacteria, as determined by APC, were 3.77 log CFU/g using the pour plate method and 3.55 log CFU/g using Petrifilm. The pairwise Student t test showed that with the pour plate TABLE 3. Comparison of recoveries for microorganisms from fresh and frozen beef using a pour plate method and Petrifilm for aerobic plate count, coliform count, and E. coli count LogCFU/ml Minimum

Maximum

Mean

APCb pour plate

0.47

7.66

3.77 ± 1.02 (n = 104)

APC Petrifilm

0.70

7.00

3.55 ± 1.02 (n = 104)

CCc pour plate

0.70

4.15

2.43 ± 0.77 (n = 77)

CC Petrifilm

0.70

4.30

2.43 ± 0.80 (n = 77)

ECCd pour plate

0.70

3.15

1.67 ± 0.80 (n = 67)

CC Petri film

0.70

3.69

2.13 ± 0.78 (n = 67)

Plating method

"Determined using pairwise Student t test. b Aerobic plate count. Coliform count. d Escherichia coli count. * Significant at the 0.05 level. C

P value"

0.0004*

0.7995

0.0001*

1087

COMPARISON OF CONVENTIONAL PLATINGANDPETRIFILM method a significantly higher number of aerobic mesophilic bacteria was recovered compared to the Petri film method (P :::; 0.05). For these samples, the mean APC was 0.22 log higher when using the pour plating technique. Similar findings have been reported by Smith et al. (6). They observed a mean count 0.25 log higher using pour plates versus Petrifilm. It is unclear why higher recoveries for mesophilic aerobic bacteria have been observed using the pour plate method. Perhaps certain mesophilic bacteria, inherently present in beef, are better resuscitated in conventional microbial media due to a more optimal water activity, oxidation/reduction potential, etc. Furthermore, APC Petrifilm uses a "Standard Methods" medium as the microbiological base, whereas our pour plating method utilized a Trypticase soy medium as the microbiological base. The Trypticase soy medium contains nearly double the carbohydrate level present in the "Standard Methods" medium. This may have provided a more suitable environment for recovering aerobic mesophilic bacteria. Current studies are being conducted in our laboratory to compare (a) pour plating using "Standard Methods" agar versus APC Petrifilm and (b) pour plating using Trypticase soy agar versus APC Petrifilm supplemented with additional carbohydrate sources. Further research efforts need to be extended to determined why these differences exist. Of the total of 46 fresh samples tested, 26 were tested for coliforms and presumptive E. coli. Of these samples, 13 (50%) contained coliforms and 12 (46%) were positive for presumptive E. coli. Of the 58 frozen products sampled, 51 (88%) contained counts for coliform bacteria and 45 (78%) were positive for presumptive E. coli. For coliforms, no differences (P > 0.05) in recovery were observed when using either the pour plate method or Petrifilm (Table 3). Average coliform counts were approximately 2.43 log CFU per gram of food using either method. When beef samples contained coliform bacteria, coliform counts were generally about 1 log less than mesophilic bacterial counts. In many cases higher recovery was observed for presumptive E. coli when Petrifilm was used as the plating medium. Presumptive E. coli counts were significantly higher using the Petrifilm plating method (P :::; 0.05) with a mean difference of 0.46 log (Table 3). For those samples that were positive for presumptive E. coli, counts were generally 1 log less than coliform counts. The data indicate that the pour plate method used in this study is better for recovery of aerobic mesophilic bacteria from beef samples. Petrifilm appears to be an equivalent method for recovery of coliform bacteria from beef samples. Furthermore, Petrifilm may have a slight advantage for recovery of E. coli. There were several samples from which E. coli could be recovered using Petri film but could not be recovered using the pour plate technique. These results are consistent with observations made by Ingham and Moody (3). A total of 56 different product surface samples and food contact surfaces were tested (Table 4). No significant differences (P > 0.05) were noted for recovery of mesophilic bacteria using the pour plate or Petri film. Average counts for mesophilic bacteria on surfaces of beef were 2.31 log CFU/cm2 and 2.22 log CFU/cm2 using the pour plate

TABLE 4. Comparison of recovery for mesophilic bacteria from food surfaces and food contact surfaces using a pour plate method and Petrifilm

LogCFU/ml Platingmethod

Minimum Maximum

Mean

APCb pour plate

0.48

5.26

2.31 ± 1.22 (n = 56)

APC Petrifilm

0.30

5.264.64

2.22 ± 1.00 (n = 56)

a b

P

valuea

0.8603

Determined using pairwise Student t test. Aerobic plate count.

method and Petri film, respectively. Food surfaces were also tested for the presence of coliform bacteria and for E. coli. When the surface swab procedure was used, bacteria from either group were seldom recovered. The low sample number for tests of recovery of coliforms and E. coli made a comparison of pour plating and Petrifilm impossible. For food contact surfaces, mesophilic bacterial levels were less than 1 CFU/cm2 immediately after sanitation and before processing. After processing began, these levels quickly rose to greater than 100 CFU/cm2• Surface bacterial levels generally remained constant thereafter. For this particular processing plant, microbial contamination of beef in contact with contaminated processing equipment is difficult to prevent or control. The average total microorganism levels inherent to the raw product were typically 1 to 1.5 log higher than the average food contact surface microbial counts (compare Tables 3 and 4). Therefore, food contact surface contamination will probably not cause a significant change in the overall microbial level of the finished product. As a result, product shelf life would likely remain unchanged. However, the presence of foodborne pathogens (e.g., E. coli 0157:H7 and Salmonella spp.) on food contact surfaces and their subsequent transfer to food are a concern. Pathogenic microorganisms present at low levels could be transferred from food contact surfaces to processed food throughout the processing day. One question that has been debated in past years is the use of buffers for recovery of surface bacteria using the surface swab technique. Common buffers used for environmental surface sampling are neutralizing buffer or letheen broth (4, 5, 8). These buffers are used to counteract (or "neutralize") the possible residual bactericidal effects of sanitizers from a swabbed surface. To study differences between these two buffers, a comparison of recovery of mesophilic bacteria from swabbed surfaces using neutralizing buffer and letheen broth was done for 26 naturally contaminated food contact surface samples. The comparison for bacterial counts was made for the pour plating technique and for plating onto Petrifilm, independently. Recoveries of surface bacteria for samples collected using letheen broth were significantly higher (P:::; 0.05) compared to those collected using neutralizing buffer according to counts obtained with either plating medium (Table 5). Furthermore, when neutralizing buffer was used for Petrifilm plating, counting was much more difficult (i.e., colonies were not

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LINTON,

EISEL, AND MURIANA

TABLE 5. Comparison of recovery of surface microorganisms using neutralizing buffer or letheen broth followed by pour plating and Petrifilm plating LogCFU/ml Plating method

Minimum

Maximum

Mean

APCb pour plate with neutralizing buffer

0.30

4.40

1.56 ± 1.29 (n=26)

APC pour plate with letheen buffer

0.30

4.32

1.77 ± 0.92 (n = 26)

APC Petrifilm with neutralizing buffer

0.48

5.08

1.82 ± 1.22 (n = 26)

APC Petrifilm with letheen buffer

0.30

5.46

2.02 ± 1.12

P valuea

0.0022*

when using Petrifilm. The only noticeable limitation in using Petrifilm is for recovery of mesophilic bacteria from beef samples. Recoveries were lower using Petrifilm. The benefits of using Petrifilm far outweigh the limitations. In addition to comparable recoveries of bacteria, Petrifilm offers many other advantages compared to pour plating for microbial analysis including no required media preparation, smaller size, easier counting of colonies, etc. The use of Petrifilm products in microbial sampling plans may be suitable for companies considering developing an in-house microbial sampling plan. REFERENCES 1.

(n

=

0.0222*

26)

Determined using pairwise Student t test. b Aerobic plate count.

a

2.

3.

distinct) and time consuming. Apparently, the neutralizing buffer interacts with and disrupts the gel matrix of Petrifilm.

4.

CONCLUSION

5.

This study has demonstrated that Petrifilm plating offers a good alternative compared to conventional plating for recovery of mesophilic bacteria, coliform bacteria, and E. coli. It is a useful method for recovery of bacteria from beef product samples, from food product surface samples, and from food contact surfaces. In most cases, Petrifilm was equivalent to or better than conventional pour plating techniques. No differences were noted for recovery of coliform bacteria, and recovery of E. coli was often better

6.

7. 8.

Chain, V. S., and D. Y. C. Fung. 1991. Comparison of Redigel®, Petrifilm®, spiral plate system, Isogrid®, and aerobic plate count for determining the number of aerobic bacteria in selected foods. J. Food Prot. 54:208-211. Federal Register. 1996. Pathogen reduction: hazard analysis and critical control point (HACCP) systems, final rule. Fed. Regist. 61:38805-38855. Ingham, S. C., and M. W. Moody. 1990. Enumeration of aerobic plate count and E. coli during blue crab processing by standard methods, Petrifilm®, and Redigel®. J. Food Prot. 53:423-424. Marriott, N. G., R. A. Garcia, and D. R. Lee. 1978. Comparison of bacterial swab samples given different storage treatments. J. Food Prot. 41:897-898. McGoldrick, K. F., T. L. Fox, and J. S. McAllister. 1986. Evaluation of a dry medium for detecting contamination on surfaces. Food Techno!. 40(4):77-80. Smith, L. B., T. L. Fox, and F. F. Busta. 1985. Comparison of a dry medium culture plate (Petrifilm® SM plates) to the aerobic plate count method for enumeration of mesophilic aerobic colony forming units in fresh ground beef. J. Food Prot. 48:1044-1045. Tompkin, R. B. 1983. Indicator organisms in meat and poultry products. Food Techno!. 37(6):107-110. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium of methods for the microbiological examination of foods, 3rd ed., p. 25-49, 57-59, 75-95, 325-369. American Public Health Association, Washington, D.C.