Enumeration of total coliforms and E. coli in foods by the SimPlate coliform and E. coli color indicator method and conventional culture methods: collaborative study

The relative effectiveness of the SimPlate Coliform and E. coli Color Indicator (CEc-CI) method was compared to the AOAC 3-tube Most Probable Number (MPN) methods for enumerating and confirming coliforms and Escherichia coli in foods (966.23 and 966.24). In this study, test portions were prepared and analyzed according to the conditions stated in both the AOAC methods and SimPlate directions for use. Six food types were artificially contaminated with coliform bacteria and E. coli: frozen burritos, frozen broccoli, fluid pasteurized milk, whole almond nut meats, cheese, and powdered cake mix. Method comparisons were conducted. Overall, the SimPlate method demonstrated <0.3 log difference for total coliform and E. coli counts compared to the AOAC reference methods for the majority of food types and levels analyzed. In all cases, the repeatability and reproducibility of the SimPlate CEc-CI method were not different from those of the reference methods and in certain cases, were statistically better than those of the AOAC 3-tube MPN methods. These results indicate that the SimPlate CEc-CI method and the reference culture methods are comparable for enumeration of both total coliforms and E. coli in foods.     

Dry rehydratable film method for enumerating confirmed Escherichia coli in poultry, meats, and seafood: collaborative study

A rehydratable dry-film plating method for Escherichia coli, the Petrifilm E. coli/Coliform (EC) Count Plate in foods, has been compared with the AOAC INTERNATIONAL most probable number (MPN) method. Eleven laboratories participated in the collaborative study. Three E. coli levels in 8 samples each of frozen raw ground turkey, frozen raw ground beef, and frozen cooked fish were tested in duplicate. Mean log counts for the Petrifilm plate procedure were not significantly different from those for the MPN procedure for cooked fish samples inoculated with low or high inocula levels, for samples of raw turkey inoculated at medium level, and for beef inoculated at low, medium, and high levels. Repeatability and reproducibility variances of the Petrifilm EC Plate method recorded at 24 h were as good as or better than those of the MPN method. The dry rehydratable film method for enumerating confirmed E. coli in poultry, meats, and seafood has been adopted first action by AOAC INTERNATIONAL.     

Detection of Salmonella in fresh cheese,poultry products,and dried egg products by the ISO 6579 Salmonella culture procedure and the AOAC official method: collaborative study

Three food types were analyzed for the presence of Salmonella by the AOAC culture method and by the International Organization for Standardization (ISO 6579:2002) culture method. Paired test portions of each food type were simultaneously analyzed by both methods. A total of 21 laboratories representing federal government agencies and private industry, in the United States and Europe, participated in this interlaboratory study. Foods were artificially contaminated with Salmonella and competing microflora if naturally contaminated sources were not available. No statistical differences (p < 0.05) were observed between the AOAC and ISO culture methods for fresh cheese and dried egg products. A statistically significant difference was observed for one of the 2 lots of poultry from the first trial. The poultry meat used in this run was radiation sterilized, artificially contaminated with Salmonella and competitive flora, and then lyophilized. A second trial was conducted with 2 separate lots of raw ground chicken that were naturally contaminated. The results from the second trial showed no statistical difference between the 2 culture methods. A third trial involving 4 laboratories was conducted on 2 separate lots of naturally contaminated raw poultry. Again, no statistically significant differences occurred. It is recommended that ISO 6579:2002 culture method for Salmonella be adopted Official First Action for the analysis of fresh cheese, fresh chilled and frozen poultry, and dried egg products.     

Evaluation of the Sanita-kun coliforms, a dehydrated medium sheet for coliform detection. Performance-Tested Method 100402

The Sanita-kun Coliforms consists of a transparent cover film, an adhesive sheet, a layer of nonwoven fabric, and a water-soluble compound film, including a culture medium formula for the detection of coliforms. The medium sheet was validated with 26 food types belonging to 9 food categories (meat, poultry, fish and seafood, fruits and vegetable, dairy, chocolate or bakery, animal feeds, pasta, and miscellaneous) using violet red bile (VRB) agar method in the U.S. Food and Drug Administration's Bacteriological Analytical Manual as a reference according to the AOAC guideline. The medium sheet showed 100% inclusivity and exclusivity. Ruggedness study suggested allowances in the incubation temperature and time as 33-35 degrees C and 24 +/- 4 h, respectively. The performance of 3 different lots of the medium sheets was equivalent and suggested no change of the performance at least for 3 years. In the comparative recovery study, many samples (84.6%), which were inoculated with a coliform strain, showed no significant difference between the 2 methods. The linear correlation coefficient (r2) to the VRB agar was calculated as 0.94. In the repeatability study, the average relative standard deviation of total foods was 0.10 in the medium sheet. In the independent study, the medium sheet detected significantly more colonies than VRB plates in the frozen raw milk sample, while there was no significant difference between the 2 methods in raw ground beef sample. Comparative recovery study on foods, inoculated and then frozen, showed the medium sheet detected injured cells with better recovery than VRB agar. The analysts in the independent study wrote that the medium sheet was easy to use and read overall. The Sanita-kun sheet provides an alternative method to coliform count agar.     

Equivalence of Visual Immunoprecipitate Assay (VIP) for Salmonella for the detection of motile and nonmotile Salmonella in all foods to AOAC culture method: collaborative study

Six foods representative of a wide variety of processed, dried powder processed, and raw food types were analyzed by the Visual Immunoprecipitate Assay (VIP) for Salmonella and AOAC INTERNATIONAL culture method. Paired samples of each food type were simultaneously analyzed; one sample by the VIP method and one by the AOAC culture method. A total of 24 laboratories representing federal government agencies and private industry, in the United States and Canada, participated in this collaborative study. Food types were inoculated with species of Salmonella with the exception of raw ground chicken, which was naturally contaminated. No statistical differences (p < 0.05) were observed between VIP for Salmonella interpretation and the AOAC culture method for any inoculation level of any food type or naturally contaminated food. The method was adopted Official First Action status by AOAC INTERNATIONAL.     

Modification of enrichment protocols for TECRA Listeria Visual Immunoassay method 995.22: collaborative study

A collaborative study was conducted to validate new enrichment methods for the TECRA Listeria Visual Immunoassay (TLVIA). These new methods incorporate a newly formulated medium, TECRA Listeria Enrichrment Broth, which does not contain the highly toxic antifungal agent, cycloheximide. The new procedures will provide an alternative to the enrichment procedures described in AOAC Method 995.22. Three food types (raw ground beef, lettuce, and ice cream) were analyzed in the United States, and 2 food types (cooked turkey and cooked fish fillets) were analyzed in Australasia. Thirty collaborators participated in the study, 16 in Australasia and 14 in the United States. With the exception of one batch of ground beef, comparison of the proportion of positive test portions (p > or = 0.05) showed no significant difference between the TLVIA and the reference method for the 5 foods at 3 inoculation levels. For the one batch of naturally contaminated raw ground beef, the TLVIA gave significantly more confirmed positive results than the reference method.     

Comparison of the compact dry EC with the most probable number method (AOAC official method 966.24) for enumeration of Escherichia coli and coliform bacteria in raw meats. Performance-Tested Method 110402

Compact Dry E. coli/Coliform Count (EC) is a ready-to-use test method for the enumeration of Escherichia coli and coliform bacteria in food. The plates are presterilized and contain culture medium and a cold water-soluble gelling agent. The medium should be rehydrated with 1 mL diluted sample inoculated onto the center of the self-diffusible medium, allowing the solution to diffuse by capillary action. The plate can be incubated at 35 degrees C for 20-24 h and the colonies counted without any further working steps. The Compact Dry EC medium plates were validated as an analysis tool for determining colony-forming units (CFU) of E. coli and coliform bacteria from a variety of raw meats using 5 different types of raw meats. The performance tests were conducted at 35 degrees C. In all studies performed, no apparent differences were observed between the Compact Dry EC method and the AOAC Official Method 966.24 results. For the accuracy claim (n = 75), a correlation factor of r2 = 0.93 (E. coli) and r2 = 0.93 (coliform bacteria) could be assigned, as stated in the application for "Performance-Tested Method."     

Equivalence of assurance Gold Enzyme Immunoassay for visual or instrumental detection of motile and nonmotile Salmonella in all foods to AOAC culture method: collaborative study

Six foods representative of a wide variety of processed, dried powder processed, and raw food types were analyzed by the Assurance Gold Salmonella Enzyme Immunoassay (EIA) and AOAC INTERNATIONAL culture method. Paired samples of each food type were simultaneously analyzed; one sample by the Assurance method and one by the AOAC culture method. The results for Assurance method were read visually and instrumentally with a microplate reader. A total of 24 laboratories representing federal government agencies and private industry, in the United States and Canada, participated in this collaborative study. Food types were inoculated with species of Salmonella with the exception of raw ground chicken, which was naturally contaminated. No statistical differences (p < 0.05) were observed between Assurance Gold Salmonella EIA with either visual or instrumental interpretation and the AOAC culture method for any inoculation level of any food type or naturally contaminated food. The Assurance visual and instrumental options of reading sample reactions produced the same results for 1277 of the 1296 sample and controls analyzed.     

Reveal 8-Hour Test System for detection of Escherichia coli O157:H7 in raw ground beef, raw beef cubes, and iceberg lettuce rinse: collaborative study.

Five different food types were analyzed by the Reveal for E. coli O157:H7 8-Hour Test System (Reveal 8) and either the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) culture method or the U.S. Department of Agriculture Food Safety Inspection Service (FSIS) culture method for the presence of E. coli O157:H7. A total of 27 laboratories representing academia and private industry in the United States and Canada participated. Food types were inoculated with E. coli O157:H7 at 2 different levels: a high level where predominantly positive results were expected, and a low level where fractional recovery was anticipated. During this study, 1,110 samples and controls were analyzed by both the Reveal 8 and by BAM or FSIS by each of the collaborators (2,220 samples in total). For each set of samples, 740 were artificially inoculated with E. coli O157:H7, and 370 were uninoculated controls. The Reveal 8 detected 528 presumptive positives of which 487 were confirmed positive by the BAM culture method. In comparison, BAM and FSIS detected 489 of the 740 artificially contaminated samples as positive. In an additional in-house study performed only on chilled and frozen raw ground beef, 240 artificially inoculated samples were analyzed by both the Reveal 8 and by FSIS. The Reveal 8 detected and confirmed 104 samples as positive compared to 79 confirmed positive by FSIS.     

Enumeration of total yeasts and molds in foods by the SimPlate Yeast and Mold-Color Indicator method and conventional culture methods: collaborative study

The relative effectiveness of the SimPlate Yeast and Mold-Color Indicator method (Y&M-CI) was compared to the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM) method and the proposed International Organization for Standardization (ISO) method, ISO/CD 21527, for enumerating yeasts and molds in foods. Test portions were prepared and incubated according to the conditions stated in both the BAM and ISO methods. Six food types were analyzed: frozen corn dogs, nut meats, frozen fruits, cake mix, cereal, and fresh cheese. Nut meats, frozen fruits, and fresh cheese were naturally contaminated. All other foods were artificially contaminated with either a yeast or mold. Seventeen laboratories throughout North America and Europe participated in the study. Three method comparisons were conducted. In general, there was <0.3 mean log count difference in recovery between the SimPlate method and the 2 corresponding reference methods. Moreover, mean log counts between the 2 reference methods were also very similar. The repeatability (Sr) and reproducibility (SR) standard deviations were comparable between the 3 method comparisons. These results indicate that the BAM method and the SimPlate method are equivalent for enumerating yeast and mold populations in foods. Similarly, the SimPlate method is comparable to the proposed ISO method when test portions are prepared and incubated as defined in the proposed ISO method.     

Evaluation of a dry, rehydratable film method for rapid enumeration of Staphylococcus aureus

Results with the new 3M Petrifilm Rapid S. aureus Count (RSA) Plate method were compared with those of the classical Baird-Parker agar (BPA) method for detection and enumeration of Staphylococcus aureus. Studies on 219 bacterial strains demonstrated that the Petrifilm RSA plate is more sensitive than and as specific as the classical BPA method for confirmed identification of S. aureus. Counts of colonies from 71 pure cultures, 61 naturally contaminated food samples, and more than 750 artificially inoculated food samples showed that the Petrifilm RSA method was as effective as the classical BPA method for identification and enumeration of S. aureus. The Petrifilm RSA method gave results in one-third the time required for the classical method.     

Rapid methods to enumerate Escherichia coli in foods using 4-methylumbelliferyl-beta-D-glucuronide

Three methods to enumerate Escherichia coli in food were compared. They were based on AOAC methods using lauryl tryptose broth (LST) medium, LST-4-methylumbelliferyl-beta-D-glucuronide (MUG) medium, and a proposed method using regular LST in combination with E. coli (EC)-MUG medium. An efficacious and cost-effective method is needed that can detect E. coli and does not produce false presumptive positives. We tested 170 cheeses, 40 frozen processed seafood samples, 210 tree nuts, and 40 other samples. The method of choice for enumerating E. coli depends on the commodity itself. For a product, such as hard cheese or processed seafood, with a history of being negative for E. coli and other lactose-fermenting organisms, the proposed method using regular LST/EC-MUG is a good choice. These samples were seldom presumptive positive in the primary LST medium. If gas was produced, EC-MUG was an effective secondary medium. No false positives (fluorescence) or negatives were detected in EC-MUG medium. For a product with a history of being positive for E. coli and/or other lactose fermenting organisms, such as tree nutmeats or cheeses that are ripened by bacteria or mold, the method using LST-MUG is the method of choice. A presumptive positive in the LST-MUG medium was highly correlative with the biochemical tests that confirmed a sample contain E. coli. For samples spiked with E. coli, the results from each of these 3 methods were identical, and were consistent in enumerating E. coli.     

Evaluation of the GeneQuence DNA hybridization method in conjunction with 24-hour enrichment protocols for detection of Salmonella spp. in select foods: collaborative study

A multilaboratory study was conducted to compare performance of the GeneQuence DNA hybridization (DNAH) method incorporating new 24 h enrichment protocols and reference culture procedures for detection of Salmonella spp. in select foods. Six food types (raw ground turkey, raw ground beef, dried whole egg, milk chocolate, walnuts, and dry pet food) were tested by the DNAH method and by the culture methods of either the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) or the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA/BAM). Fifteen laboratories participated in the study. Four of the foods tested (raw ground turkey, dried whole egg, milk chocolate, and dry pet food), showed no statistically significant differences in performance between the DNAH method and the reference procedure as determined by Chi square analysis. Sensitivity rates for the DNAH method ranged from 92 to 100%. The DNAH method, with the specific enrichment protocol evaluated, was found to be ineffective for detection of Salmonella spp. in walnuts. For raw ground beef, results from one trial showed a statistically significant difference in performance, with more positives obtained by the reference method. However, evidence suggests that the difference in the number of positives was likely due to lack of homogeneity of the test samples rather than to DNAH method performance.     

Evaluation of VIDAS Listeria species Xpress (LSX) immunoassay method for the detection of Listeria species in foods: collaborative study

In a multilaboratory study, the effectiveness of an alternative method for rapid screening of Listeria species compared to traditional reference methods was demonstrated in a variety of food products. A collaborative study was conducted to compare the VIDAS Listeria species Xpress (LSX) method and the standard cultural methods for the detection of Listeria species in foods. Six food types were tested: vanilla ice cream, cheddar cheese, raw ground beef, frozen green beans, deli turkey, and cooked shrimp. Each food, inoculated with a different Listeria strain at two levels and uninoculated test portions, was analyzed by each method. A total of 15 laboratories representing government and industry participated. In this study 1134 tests were analyzed in the statistical analysis. There were 490 positives by the VIDAS LSX method using the sample boiling step, 483 positives by the VIDAS LSX method using the Heat and Go system, and 439 positives by the standard culture methods. Overall, the Chi-square result for the VIDAS LSX method with boiling for all foods was 7.25, indicating a significant statistical difference between the VIDAS method and the standard methods at the 5% confidence. For the VIDAS LSX method with the Heat and Go system, the Chi-square result for all foods was 5.37, indicating a significant statistical difference between the VIDAS LSX assay with the Heat and Go system and the standard methods at the 5% level of significance. In both cases, the VIDAS method was more sensitive than the standard methods. The LSX method detects Listeria species in foods with negative or presumptive positive results in a minimum of 30 h compared to at least 5 days for the cultural methods. Based on the results of this collaborative study, it is recommended that the VIDAS LSX method be adopted as an AOAC Official Method for the detection of Listeria species in dairy products, vegetables, seafood, raw meats and poultry, and processed meats and poultry.     

Evaluation of the BAX system for detection of Salmonella in selected foods: collaborative study

A multilaboratory study was conducted to compare the automated BAX System to the standard cultural methods for detection of Salmonella in selected foods. Five food types--frankfurters, raw ground beef, mozzarella cheese, raw frozen tilapia fish, and orange juice--at 3 inoculation levels, were analyzed by each method. A sixth food type, raw ground chicken, was tested using 3 naturally contaminated lots. A total of 16 laboratories representing government and industry participated. In this study, 1386 samples were analyzed, of which 1188 were paired samples and 198 were unpaired samples. Of the 1188 paired samples, 461 were positive by both methods and 404 were negative by both methods. Thirty-seven samples were positive by the BAX System but negative by the standard reference method, and 11 samples were positive by standard cultural method and negative by the BAX System. Of the 198 unpaired samples, 106 were positive by the BAX System and 60 were positive by the standard cultural method. A Chi square analysis of each of the 6 food types, at the 3 inoculation levels tested, was performed. For all foods, the BAX System demonstrated results comparable to those of the standard reference methods based on the Chi square results.     

Two rapid methods for detection of Escherichia coli exceeding 10(4)/g action levels: precollaborative study

The current AOAC Method 966.24 for enumeration of Escherichia coli in foods uses a most probable number (MPN) procedure with extensive confirmation steps. Two new methods based on membrane filtration (MF) were compared to the MPN reference method for detection of high levels of E. coli in 5 food types, some of which represent categories for which the U.S. Food and Drug Administration (FDA) mandates additional testing if an action level of 10(4)/g E. coli is exceeded. Ground beef, which is not FDA regulated, was also tested. The 5 food types were all inoculated at 3 levels: 10(2)/g, > or = 10(4)/g, and > or = 10(5)/g E. coli. An MF protocol using either m-ColiBlue24 (CB) or lauryl sulfate tryptose plus BCIG (LST/BCIG) was an effective potential alternative to the reference method. Sensitivity and specificity for both CB and LST/BCIG were 98 and 100%, respectively. Agreement between MPN and both CB and LST/BCIG was 98%. The 2 proposed methods allow completion of both presumptive and confirmatory steps in 1-3 days, whereas the reference method requires as many as 11 days. Exclusivity testing with 50 non-E. coli strains indicated 100% were correctly ruled out by the proposed protocols. Inclusivity testing was used to determine whether typical results were obtained after incubation of E. coli cultures on CB or LST/BCIG for 24 h. Of 50 E. coli strains tested, 100% yielded typical results after incubation on CB, and 98% yielded typical results after incubation on LST/BCIG.     

Validation of the Soleris E. coli method for detection and semi-quantitative determination of Escherichia coli in foods

The performance of the Soleris E. coli method was compared with that of the ISO 7251 most probable number (MPN) and detection reference methods for Escherichia coli. The Soleris E. coli method is a growth-based, rapid, automated system composed of temperature-controlled incubation chambers and photodiode-based optical detection devices for measurement of color changes in a prepared medium vial. A dilution of the test sample homogenate is inoculated directly into the vial. Products of E. coli metabolism alter the color of the medium over time, and this change is monitored by the Soleris instrument. The test is used in a dilute-to-specification or specification monitoring manner in which the result is positive or negative around a desired cutoff (in CFU/g) determined by the dilution and volume of sample homogenate added to the vial. Alternatively, the test is used for zero tolerance determinations (e.g., absence in 25 g) by performing an off-line pre-enrichment step followed by transfer of a portion of the pre-enrichment culture to the Soleris vial. Six E. coli strains originating from food sources were inoculated individually into six food commodities: frozen green beans, Echinacea powder, cocoa powder, sweetened condensed milk, pasteurized liquid egg, and shredded mozzarella cheese. Uninoculated samples were included in each trial. The results obtained by the ISO 7251 detection method and the Soleris E. coli method were shown to be in agreement by Chi-square analysis when the presence of E. coli was determined in 25 g of sample. Results from the Soleris E. coli dilute-to-specification method and the ISO 7251 MPN method were found to be in agreement by probability of detection statistical analysis. In inclusivity testing, 52 of 53 E. coli strains were detected within 24 h. Only a non-thermoduric strain of serotype O157:H43 was not detected. In exclusivity testing, all 31 strains tested produced negative results. Results of ruggedness experiments show that accurate results can be obtained even when the operating temperature of the Soleris instrument is set beyond normal tolerances. The internal and independent laboratory studies demonstrated that the Soleris E. coli method could be successfully utilized as an alternative to the reference methods, with a significant time savings of 2 to 3 days.     

Validation of RAPID'Staph for enumeration of Staphylococcus aureus in foods. Performance-Tested Method

RAPID'Staph (Bio-Rad Laboratories, Hercules, CA) is a medium for differentiation and enumeration of coagulase-positive Staphylococcus aureus in food. RAPID'Staph medium is based on a Baird Parker formula optimized for the detection and enumeration of S. aureus in 24 h. The principle of the medium relies on the capacity of S. aureus to reduce tellurite (production of black colonies) and to provoke proteolysis of egg yolk (production of clear halo around the colony). Four foods (pasteurized whole milk, custard pie, processed ham, and smoked salmon) were selected to compare the performance of RAPID'Staph agar to AOAC Official Method 975.55. Method comparison studies demonstrated excellent agreement between the methods. Inclusivity and exclusivity rates of the medium were 100%. RAPID'Staph agar performed as expected when minor procedural variations were introduced, validating the ruggedness of the method. There was no difference in performance between the dehydrated and ready-to-use formulations of the media.     

Evaluation of VIDAS Salmonella (SLM) easy Salmonella method for the detection of Salmonella in a variety of foods: collaborative study

The VIDAS Salmonella (SLM) Easy Salmonella method is a specific enzyme-linked fluorescent immunoassay performed in the automated VIDAS instrument. The VIDAS Easy Salmonella method is a simple 2-step enrichment procedure, using pre-enrichment followed by selective enrichment in a newly formulated broth, SX2 broth. This new method was compared in a multilaboratory collaborative study to the U.S. Food and Drug Administration's Bacteriological Analytical Manual, Chapter 5 method for five food matrixes (liquid egg, vanilla ice cream, spinach, raw shrimp, and peanut butter) and the U.S. Department of Agriculture's Microbiology Laboratory Guidebook 4.04 method for deli turkey. Each food type was artificially contaminated with Salmonella at three inoculation levels. A total of 15 laboratories representing government, academia, and industry, throughout the United States, participated. In this study, 1583 samples were analyzed, of which 792 were paired replicates and 791 were unpaired replicates. Of the 792 paired replicates, 285 were positive by both the VIDAS and reference methods. Of the 791 unpaired replicates, 341 were positive by the VIDAS method and 325 were positive by the cultural reference method. A Chi-square analysis of each of the six food types was performed at the three inoculation levels tested. For all foods evaluated, the VIDAS Easy SLM method demonstrated results comparable to those of the reference methods for the detection of Salmonella.     

Petrifilm rapid S. aureus Count Plate method for rapid enumeration of Staphylococcus aureus in selected foods: collaborative study

A rehydratable dry-film plating method for Staphylococcus aureus in foods, the 3M Petrifilm Rapid S. aureus Count Plate method, was compared with AOAC Official Method 975.55 (Staphylococcus aureus in Foods). Nine foods-instant nonfat dried milk, dry seasoned vegetable coating, frozen hash browns, frozen cooked chicken patty, frozen ground raw pork, shredded cheddar cheese, fresh green beans, pasta filled with beef and cheese, and egg custard-were analyzed for S. aureus by 13 collaborating laboratories. For each food tested, the collaborators received 8 blind test samples consisting of a control sample and 3 levels of inoculated test sample, each in duplicate. The mean log counts for the methods were comparable for pasta filled with beef and cheese; frozen hash browns; cooked chicken patty; egg custard; frozen ground raw pork; and instant nonfat dried milk. The repeatability and reproducibility variances of the Petrifilm Rapid S. aureus Count Plate method were similar to those of the standard method.