Comparison of Conventional Plating Methods and Petrifilm for the Recovery of Microorganisms in a Ground Beef Processing Facilityt

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R. H. LINTON, * W. G. EISEL, and P. M. MURIANA Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/9/1084/1665132/0362-028x-60_9_1084.pdf by guest on 27 September 2021

Department of Food Science, 1160 Smith Hall, Purdue University, West Lafayette, IN 47907, USA

(MS# 96-240: Received 10 September 1996/Accepted 2 December 1996)

ABSTRACT practices (GMPs), respectively. HACCP and GMP programs have been used for over 20 years in food processing plants. The objective of this study was to compare recovery of More recently, HACCP programs have been broadened to microorganisms for various beef samples and beef contact surfaces cover the entire food production chain-from the farm to the using conventional pour plating techniques and Petri film methods. plate of consumers. Increased emphasis for HACCP pro- Comparisons for aerobic plate count (APC), coliform count (CC), gram development is being directed toward farm, slaughter, and Escherichia coli count (ECC) were done for 104 fresh or frozen retail cuts and 56 food surface or food contact surfaces. and harvest operations. An integral component of HACCP Samples were taken at a midwestern retail ground beef processing programs is periodic testing of microbial contamination plant during a 12-month project. APC comparisons were made for levels. Common sampling protocols include microbial assess- pour plating using Trypticase soy agar versus Aerobic Plate Count ment from products at various steps in the food process, Petrifilm. CC and ECC were compared for pour plating using violet from food contact surfaces, from water, and from air. red bile + MUG agar versus E. coli Petrifilm. Overall, paired t tests Microbial sampling plans differ greatly depending on the revealed a significantly higher recovery for APC from fresh and food product and the food process. frozen beef samples using the pour plating technique (P :S 0.05). Current legislation has been published by the United No significant differences (P > 0.05) were observed for CC from States Department of Agriculture Food Safety and Inspec- fresh and frozen meat samples. Recovery of E. coli from many beef samples was better using Petrifilm. Significantly higher ECCs were tion Service (USDA-FSIS) which includes several changes observed from fresh and frozen meat samples using Petrifilm to the meat and poultry inspection program (2). Under the compared to the pour plating technique (P:S 0.05). For food new legislation, meat and poultry plants will be required to surfaces and food contact surfaces, a comparison between pour adopt and carry out HACCP programs. HACCP programs plating and Petrifilm was done for aerobic plate count. No will be continually monitored by USDA inspectors. Manda- significant differences (P > 0.05) in recovery could be found tory testing for E. coli will also be required six months after between methods. A comparison between neutralizing buffer and the final rule for establishments that slaughter meat and letheen broth for recovery of surface microorganisms was done for poultry. The required frequency of E. coli testing will be both the APC pour plating method and APC Petrifilm. In both based on production volume. E. coli testing for poultry cases, recovery when using letheen broth was significantly products will be done using a carcass rinse, whereas (P :S 0.05) higher than neutralizing buffer. Because it is convenient and gave comparative results, Petrifilm offers a good alternative for carcasses of beef and pork will be sampled using a surface environmental microbial testing and red meat product testing. sponge technique. The rationale for testing for E. coli is that the E. coli results may provide useful information regarding Key words: Pour-plate, Petrifilm, coliform, Escherichia coli plant sanitation, potential presence of other enteric bacteria, and process control. Sanitation standard operating proce- dures (SSOPs) will also be required as part of the regulations. In recent years, there has been a growing concern To establish these new programs, a microbial sampling regarding the safety and quality of meat and poultry plan for meat and poultry slaughter and processing plants products. The most recognized approach for controlling would likely include periodic monitoring of aerobic meso- food safety and quality is to establish hazard analysis critical philic bacteria, coliform bacteria, and E. coli. The number of control point (HACCP) programs and good manufacturing 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 * Author for correspondence. Tel: 765-494-6481; Fax: 765-494-7953. t Journal paper no. 15171 of Purdue University, Agricultural Research and hence the probability of cross-contamination from the Programs. food contact surface to a food. Coliform and E. coli counts COMPARISON OF CONVENTIONAL PLATING AND PETRIFILM 1085 from beef products and food contact surfaces can be used to MATERIALS AND METHODS indicate potential safety and quality. High coliform and E. coli counts suggest that other foodborne pathogens of Description of the processing plant A 12-month study was done at a midwestern ground beef fecal origin may be present. High coliform and E. coli counts processing facility. Raw material was received as carcass beef and on food contact surfaces can be used to indicate insufficient boxed beef (chuck, round, or sirloin). Carcass beef was then cleaning and sanitizing programs (7). trimmed, cut into retail cuts, packaged, and stored at refrigeration Traditional methods for the enumeration of aerobic (O°C). The trim was then combined with boxed beef and ground to bacteria, coliforms, and E. coli include sampling a food or fabricate ground beef of a desired fat level. Ground beef was food contact surface, followed by pour plating on a suitable packaged in 5-lb (2.26-kg) chubs and then held at refrigeration microbial medium (8). For in-house microbial analysis of (O°C). foods, this type of analysis may be time-consuming (i.e., requiring media preparation) and more difficult to perform. Fresh andfrozen product sampling procedure Product sampling included a microbial assessment of fresh An alternative method is the use of Petrifilm (3M Health and frozen beef samples. The term "fresh" is used to describe Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/9/1084/1665132/0362-028x-60_9_1084.pdf by guest on 27 September 2021 Care, St. Paul, MN). Petrifilm is a ready-made dehydrated samples that were taken at the plant during the processing of microbial medium contained on a paper surface covered by a ground beef. Fresh products were collected every two weeks. Fresh plastic film. The use of Petrifilm to recover microorganisms product samples were taken along the processing line from in a food plant may be more practical compared to pour incoming product to finished product to represent all areas of the plating techniques. When using Petrifilrn, no media prepara- process. For each sample, a sample weighing more than 25 g was tion is required, incubator and storage space is reduced, and aseptically taken (with a sterile spoon, knife, etc.), placed into a enumeration of colonies is facilitated because Petrifilm sterile sampling bag, and immediately placed into a mobile contains counting grids and a visible indicator dye. incubator held at 4°C. "Frozen" samples included various retail Comparisons of pour plating and Petrifilm have been beef cuts, boxed beef, and ground beef samples. These samples done for various products. Ingham and Moody (3) compared were collected by personnel working in the plant, placed into sterile bags, and held at -20°C until analysis. "Standard Methods" pour plates to Petrifilm "Standard Upon return to the laboratory, fresh product samples were Methods" films and compared a five-tube E. coli most weighed and aseptically trimmed to a sample weight of approxi- probable number (MPN) procedure to Petrifilm E. coli count mately 25 g. Each ofthe 25-g samples was then placed into a sterile films. No significant differences in aerobic bacterial counts stomacher bag, and 225 ml of 0.1 % (wt/vol) peptone buffer was (P > 0.05) were found between pour plating and Petrifilm. added to make a 10-1 dilution. Samples were further diluted in However, Petri film appeared to be a more sensitive method, 0.1 % peptone as needed, and plated in duplicate onto Trypticase compared to MPN, for detection of E. coli. Chain and Fung soy agar (TSA) and violet red bile agar (VRBA) + methylumbel- (1) compared Redigel (3M Health Care, St. Paul, MN), liferyl-l3-glucuronide (MUG) using a pour plate with overlay Petrifilm, spiral plating, and pour plating using standard technique and onto Aerobic (APC) Petrifilm and E. coli Petri film. plate count agar for several different foods (chicken, ground All media were incubated at 37°C. Coliforms and E. coli were enumerated from VRBA + MUG plates after a 24-h incubation beef, ground pork, shelled pecan, raw milk, thyme, and period. Pink or purple colonies were recorded as coliforms, and flour). The results showed that all five methods were highly those colonies that fluoresced under ultraviolet light were reported comparable (r = 0.97). as presumptive E. coli. On E. coli Petrifilm, after 24 h, coliforms Smith et al. (6) compared Petrifilm and pour plating for appeared pink, purple, or blue with gas production. The blue the enumeration of aerobic mesophilic bacteria in retail colonies with gas production were recorded as E. coli. For APCs, ground beef samples. Total colony-forming units were mesophilic aerobic bacteria were enumerated after 48 h from TSA determined for 119 retail ground beef samples during a and from APC Petrifilm. Frozen product samples were tested in the nine-week study. The mean counts using the pour plate same manner as fresh products except that frozen samples were method and Petrifilm method were 6.11 log CFU/g and 5.85 allowed to thaw overnight in a 4°C incubator prior to microbial analysis. In all cases, plate counts were determined and converted log CFU/g, respectively. Mean counts using the pour plate to log CFU/g (8). Table 1 lists beef product samples that were method were 0.25 log higher than those with Petrifilm tested. (P ~ 0.01). It is evident that more microbial sampling programs Food contact surface and product surface sampling procedure will be established in the near future for meat slaughter and For food surfaces and food contact surfaces, a surface swab meat processing plants. Whether samples are done "in- technique was used as has been described (8). A sterile polypropyl- house" or sent out to a testing laboratory, microbial samples ene template was used to sample a lO-cm2 surface area. A sterile for meat products and meat contact surfaces will require calcium alginate swab was introduced into 10 ml of sterile additional time and money. Any measures that can be taken neutralizing buffer (Difco Laboratories, Detroit, MI) or sterile 2 to reduce time and money, without disrupting the integrity of letheen buffer (Difco), a lO-cm area was sampled, and the swab placed aseptically back into the respective buffer. Immediately after HACCP programs, will be useful to meat slaughter and taking the sample, the tube containing the swab was then placed in processing operations. a mobile incubator and maintained at 4°C until arrival at the The objective of this study was to evaluate conventional laboratory. The elapsed time from collection of samples to plating pour plating techniques versus Petrifilm methods for recov- was between 1 and 2 h after the samples were placed in the mobile ery of aerobic bacteria, coli forms, and E. coli from various incubator. Samples collected with neutralizing buffer or letheen fresh or frozen beef products and for food contact surfaces in broth were used for the pour plate technique or Petrifilm plating, a beef processing facility. respectively. 1086 LINTON, EISEL, AND MURIANA

TABLE 1. Fresh and frozen beef samples collected at the ground method (8). Letheen broth has been recommended for surface swab beefprocessing plant samples when followed by plating onto Petrifilm (5). The effects of these two buffers on recovery of bacteria from surfaces were Sampling type Description of sample studied for various food surfaces and food contact surface samples. Fresh carcass beef Brisket area For each surface sample, neutralizing buffer and letheen broth were Fresh carcass beef Flank area used independently on adjacent food contact surfaces or product Fresh carcass beef Round area surfaces. Plating in each case was done for aerobic mesophilic Fresh carcass beef Skirt area bacteria only using the conventional pour plating techniques and Fresh boxed beef Cow Petrifilm. Surface samples for this comparison were the same as Fresh boxed beef Bull discussed above. Fresh ground beef Course grind Fresh ground beef Fine grind Statistical analysis Fresh ground beef Finished, packaged product Comparisons of microbial counts for traditional pour plating Fresh retail beef cuts Sirloin steak, rib eye steak, T-bone steak versus Petrifilm plating for aerobic bacteria, coliform count, and E. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/9/1084/1665132/0362-028x-60_9_1084.pdf by guest on 27 September 2021 Frozen carcass beef Cow coli were done separately for fresh and frozen product surfaces, Frozen boxed beef Cow and, for food contact surfaces and food surfaces. A comparison of Frozen boxed beef Bull microbial counts using letheen broth versus neutralizing buffer for Frozen ground beef Course grind aerobic bacterial counts was done for pour plating versus Petrifilm. Frozen ground beef Fine grind 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). Once at the laboratory, each tube was agitated on a Vortex mixer for 10 s to assure mixture of the sample. Samples were RESULTS AND DISCUSSION serially diluted as needed in 0.1 % (wt/vol) peptone buffer and plated in duplicate onto TSA and VRBA + MUG using a pour plate The beef samples that were tested included 46 different technique and onto APC Petri film and E. coli Petri film. All media fresh samples and 58 different frozen samples. Samples were incubated at 37°C and enumerated as described above. Table from both fresh and frozen beef were taken at all areas 2 lists surface samples that were tested. during production of ground beef from receipt of raw ingredients to completion of the finished product. Ranges Neutralizing buffer versus letheen broth for surface samples Neutralizing buffer is part of the recommended procedure and mean values for recoveries of mesophilic bacteria, when performing a surface swab followed by a pour plate coliform bacteria, and E. coli for the fresh and frozen samples are presented in Table 3. The average recovery values for aerobic mesophilic TABLE 2. Surface samples collected at the ground beefprocessing bacteria, as determined by APC, were 3.77 log CFU/g using plant the pour plate method and 3.55 log CFU/g using Petrifilm. Sampling location Description of sample The pairwise Student t test showed that with the pour plate

Floor Loading dock Floor Carcass cooler TABLE 3. Comparison of recoveries for microorganisms from Floor Trim cooler fresh and frozen beef using a pour plate method and Petrifilm for Floor Ground beef line aerobic plate count, coliform count, and E. coli count Floor Fabrication (cuts of beef) line LogCFU/ml Floor Retail packaging line Wall Receiving truck Plating method Minimum Maximum Mean P value" Wall Carcass cooler Wall Trim cooler APCb pour plate 0.47 7.66 3.77 ± 1.02 Wall Ground beef line (n = 104) Wall Fabrication (cuts of beef) line APC Petrifilm 0.70 7.00 3.55 ± 1.02 0.0004* Wall Retail packaging line (n = 104) Equipment, ground beef line Conveyor belt Equipment, ground beef line Stainless steel funnel CCc pour plate 0.70 4.15 2.43 ± 0.77 Equipment, ground beef line Stainless steel bins (n = 77) Equipment, ground beef line Stainless steel under conveyer belt CC Petrifilm 0.70 4.30 2.43 ± 0.80 0.7995 Fabrication (cuts of beef) line Conveyor belt (n = 77) Fabrication (cuts of beef) line Stainless steel plates ECCd pour plate 0.70 3.15 1.67 ± 0.80 Fabrication (cuts of beef) line Cutting boards (n = 67) Fabrication (cuts of beef) line Cutting knives Retail packaging line Stainless steel plates CC Petri film 0.70 3.69 2.13 ± 0.78 0.0001* Retail packaging line Cutting board (n = 67) Carcass beef Brisket area "Determined using pairwise Student t test. Carcass beef Flank area b Aerobic plate count. Carcass beef Round area C Coliform count. Carcass beef Skirt area d Escherichia coli count. Boxed beef Chuck * Significant at the 0.05 level. COMPARISONOF CONVENTIONALPLATINGANDPETRIFILM 1087 method a significantly higher number of aerobic mesophilic TABLE 4. Comparison of recovery for mesophilic bacteria from bacteria was recovered compared to the Petri film method food surfaces and food contact surfaces using a pour plate method (P :::; 0.05). For these samples, the mean APC was 0.22 log and Petrifilm higher when using the pour plating technique. Similar LogCFU/ml findings have been reported by Smith et al. (6). They observed a mean count 0.25 log higher using pour plates Platingmethod Minimum Maximum Mean P valuea versus Petrifilm. APCb pour plate 0.48 5.26 2.31 ± 1.22 It is unclear why higher recoveries for mesophilic (n = 56) aerobic bacteria have been observed using the pour plate APC Petrifilm 0.30 5.264.64 2.22 ± 1.00 0.8603 method. Perhaps certain mesophilic bacteria, inherently (n = 56) present in beef, are better resuscitated in conventional microbial media due to a more optimal water activity, a Determined using pairwise Student t test. oxidation/reduction potential, etc. Furthermore, APC Petri- b Aerobic plate count. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/9/1084/1665132/0362-028x-60_9_1084.pdf by guest on 27 September 2021 film uses a "Standard Methods" medium as the microbiological base, whereas our pour plating method utilized a Trypticase soy medium as the microbiological base. The method and Petri film, respectively. Food surfaces were also Trypticase soy medium contains nearly double the carbohy- tested for the presence of coliform bacteria and for E. coli. drate level present in the "Standard Methods" medium. This When the surface swab procedure was used, bacteria from may have provided a more suitable environment for recover- either group were seldom recovered. The low sample ing aerobic mesophilic bacteria. Current studies are being number for tests of recovery of coliforms and E. coli made a conducted in our laboratory to compare (a) pour plating comparison of pour plating and Petrifilm impossible. using "Standard Methods" agar versus APC Petrifilm and For food contact surfaces, mesophilic bacterial levels (b) pour plating using Trypticase soy agar versus APC were less than 1 CFU/cm2 immediately after sanitation and Petrifilm supplemented with additional carbohydrate sources. before processing. After processing began, these levels Further research efforts need to be extended to determined quickly rose to greater than 100 CFU/cm2• Surface bacterial why these differences exist. levels generally remained constant thereafter. For this particu- Of the total of 46 fresh samples tested, 26 were tested lar processing plant, microbial contamination of beef in for coliforms and presumptive E. coli. Of these samples, 13 contact with contaminated processing equipment is difficult (50%) contained coliforms and 12 (46%) were positive for to prevent or control. The average total microorganism presumptive E. coli. Of the 58 frozen products sampled, 51 levels inherent to the raw product were typically 1 to 1.5 log (88%) contained counts for coliform bacteria and 45 (78%) higher than the average food contact surface microbial were positive for presumptive E. coli. For coliforms, no counts (compare Tables 3 and 4). Therefore, food contact differences (P > 0.05) in recovery were observed when surface contamination will probably not cause a significant using either the pour plate method or Petrifilm (Table 3). change in the overall microbial level of the finished product. Average coliform counts were approximately 2.43 log CFU As a result, product shelf life would likely remain un- per gram of food using either method. When beef samples changed. However, the presence of foodborne pathogens contained coliform bacteria, coliform counts were generally (e.g., E. coli 0157:H7 and Salmonella spp.) on food contact about 1 log less than mesophilic bacterial counts. In many surfaces and their subsequent transfer to food are a concern. cases higher recovery was observed for presumptive E. coli Pathogenic microorganisms present at low levels could be when Petrifilm was used as the plating medium. Presump- transferred from food contact surfaces to processed food tive E. coli counts were significantly higher using the throughout the processing day. Petrifilm plating method (P :::; 0.05) with a mean difference One question that has been debated in past years is the of 0.46 log (Table 3). For those samples that were positive use of buffers for recovery of surface bacteria using the for presumptive E. coli, counts were generally 1 log less than surface swab technique. Common buffers used for environ- coliform counts. mental surface sampling are neutralizing buffer or letheen The data indicate that the pour plate method used in this broth (4, 5, 8). These buffers are used to counteract (or study is better for recovery of aerobic mesophilic bacteria "neutralize") the possible residual bactericidal effects of from beef samples. Petrifilm appears to be an equivalent sanitizers from a swabbed surface. To study differences method for recovery of coliform bacteria from beef samples. between these two buffers, a comparison of recovery of Furthermore, Petrifilm may have a slight advantage for recovery mesophilic bacteria from swabbed surfaces using neutraliz- of E. coli. There were several samples from which E. coli ing buffer and letheen broth was done for 26 naturally could be recovered using Petri film but could not be recov- contaminated food contact surface samples. The comparison ered using the pour plate technique. These results are consistent for bacterial counts was made for the pour plating technique with observations made by Ingham and Moody (3). and for plating onto Petrifilm, independently. Recoveries of A total of 56 different product surface samples and food surface bacteria for samples collected using letheen broth contact surfaces were tested (Table 4). No significant were significantly higher (P:::; 0.05) compared to those differences (P > 0.05) were noted for recovery of meso- collected using neutralizing buffer according to counts philic bacteria using the pour plate or Petri film. Average obtained with either plating medium (Table 5). Furthermore, counts for mesophilic bacteria on surfaces of beef were 2.31 when neutralizing buffer was used for Petrifilm plating, log CFU/cm2 and 2.22 log CFU/cm2 using the pour plate counting was much more difficult (i.e., colonies were not 1088 LINTON, EISEL, AND MURIANA

TABLE 5. Comparison of recovery of surface microorganisms when using Petrifilm. The only noticeable limitation in using using neutralizing buffer or letheen broth followed by pour plating Petrifilm is for recovery of mesophilic bacteria from beef and Petrifilm plating samples. Recoveries were lower using Petrifilm. LogCFU/ml The benefits of using Petrifilm far outweigh the limita- tions. In addition to comparable recoveries of bacteria, Plating method Minimum Maximum Mean P valuea Petrifilm offers many other advantages compared to pour APCb pour plate 0.30 4.40 1.56 ± 1.29 plating for microbial analysis including no required media with neutralizing (n=26) preparation, smaller size, easier counting of colonies, etc. buffer The use of Petrifilm products in microbial sampling plans APC pour plate 0.30 4.32 1.77 ± 0.92 0.0022* may be suitable for companies considering developing an with letheen (n = 26) in-house microbial sampling plan. buffer

APC Petrifilm with 0.48 5.08 1.82 ± 1.22 REFERENCES Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/9/1084/1665132/0362-028x-60_9_1084.pdf by guest on 27 September 2021 neutralizing (n = 26) buffer 1. Chain, V. S., and D. Y. C. Fung. 1991. Comparison of Redigel®, Petrifilm®, spiral plate system, Isogrid®, and aerobic plate count for APC Petrifilm with 0.30 5.46 2.02 ± 1.12 0.0222* determining the number of aerobic bacteria in selected foods. J. Food letheen buffer (n 26) = Prot. 54:208-211. 2. Federal Register. 1996. Pathogen reduction: hazard analysis and a Determined using pairwise Student t test. critical control point (HACCP) systems, final rule. Fed. Regist. b Aerobic plate count. 61:38805-38855. 3. Ingham, S. C., and M. W. Moody. 1990. Enumeration of aerobic plate count and E. coli during blue crab processing by standard methods, distinct) and time consuming. Apparently, the neutralizing Petrifilm®, and Redigel®. J. Food Prot. 53:423-424. buffer interacts with and disrupts the gel matrix of Petrifilm. 4. 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. CONCLUSION 5. McGoldrick, K. F., T. L. Fox, and J. S. McAllister. 1986. Evaluation of a dry medium for detecting contamination on surfaces. Food Techno!. This study has demonstrated that Petrifilm plating offers 40(4):77-80. a good alternative compared to conventional plating for 6. 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 recovery of mesophilic bacteria, coliform bacteria, and method for enumeration of mesophilic aerobic colony forming units in E. coli. It is a useful method for recovery of bacteria from fresh ground beef. J. Food Prot. 48:1044-1045. beef product samples, from food product surface samples, 7. Tompkin, R. B. 1983. Indicator organisms in meat and poultry and from food contact surfaces. In most cases, Petrifilm was products. Food Techno!. 37(6):107-110. 8. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium of equivalent to or better than conventional pour plating methods for the microbiological examination of foods, 3rd ed., p. techniques. No differences were noted for recovery of 25-49, 57-59, 75-95, 325-369. American Public Health Association, coliform bacteria, and recovery of E. coli was often better Washington, D.C.