Dry rehydratable film method for rapid enumeration of coliforms in foods (3M Petrifilm Rapid Coliform Count plate): collaborative study

A rehydratable dry-film plating method for coliforms in foods, the 3M Petrifilm Rapid Coliform Count plate method, was compared with the U.S. Food and Drug Administration's Bacteriological Analytical Manual method for nondairy foods and the American Public Health Association's Standard Methods for the Examination of Dairy Products (SMEDP) method for dairy foods. Six food types, vanilla ice cream, cheddar cheese, fresh refrigerated uncooked pasta, wheat flour, prepared frozen macaroni and cheese, and frozen hash browns, were analyzed for coliforms by 11 collaborating laboratories. For each food product tested, the collaborators received 8 blind samples consisting of a control sample and 3 levels of inoculated sample, each in duplicate. The mean log counts for the methods were comparable. The repeatability and reproducibility variances of the Petrifilm Rapid Coliform Count method at 14 and 24 h were not significantly different from those of the standard methods.     

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.     

3M Petrifilm Staph Express Count plate method for the enumeration of Staphylococcus aureus in selected types of processed and prepared foods: collaborative study

The 3M Petrifilm Staph Express Count plate method was compared with AOAC Official Method 975.55 for the enumeration of Staphylococcus aureus in selected foods. Five foods--frozen lasagna, custard, frozen mixed vegetables, frozen hashbrowns, and frozen batter-coated mushrooms--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, a low inoculation level, a medium inoculation level, and a medium inoculation level with background flora, each in duplicate. The mean log10 counts for the methods were comparable for all 5 foods. The repeatability and reproducibility variances of the 24 h Petrifilm Staph Express Count plate method were similar to those of the 72 h standard method.     

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.     

3M Petrifilm Staph Express Count plate method for the enumeration of Staphylococcus aureus in selected types of meat, seafood, and poultry: collaborative study

The 3M Petrifilm Staph Express Count plate method was compared with AOAC Official Method 975.55 for the enumeration of Staphylococcus aureus in selected foods. Four foods--cooked, diced chicken; cured ham; smoked salmon; and pepperoni--were analyzed for S. aureus by 12 collaborating laboratories. For each food tested, the collaborators received 8 blind test samples consisting of a control sample, a low inoculation level, a medium inoculation level, and a medium inoculation level with background flora, each in duplicate. The mean log10 counts for the methods were comparable for all 4 foods. The repeatability and reproducibility variances of the 24 h Petrifilm Staph Express Count plate method were similar to those of the 72 h standard method.     

3M Petrifilm Staph Express Count plate method for the enumeration of Staphylococcus aureus in selected dairy foods: collaborative study

The 3M Petrifilm Staph Express Count plate method was compared with AOAC Official Method 975.55 for the enumeration of Staphylococcus aureus in selected foods. Five foods--ice cream, raw milk, yogurt, whey powder, and cheese--were analyzed for S. aureus by 12 collaborating laboratories. For each food tested, the collaborators received 8 blind test samples consisting of a control sample, a low inoculation level, a medium inoculation level, and a medium inoculation level with background flora, each in duplicate. The mean log10 counts for the methods were comparable for all 5 foods. The repeatability and reproducibility variances of the 24 h Petrifilm Staph Express Count plate method were similar to those of the 72 h standard method.     

Evaluation of VIDAS listeria monocytogenes II (LMO2) immunoassay method for the detection of Listeria monocytogenes in foods: collaborative study

A multilaboratory study was conducted to compare the VIDAS Listeria monocytogenes II (LMO2) immunoassay and the standard cultural methods for the detection of Listeria monocytogenes in foods. Five food types-vanilla ice cream, brie cheese, cooked roast beef, frozen green beans, and frozen tilapia fish-at 3 levels were analyzed by each method. A total of 26 laboratories representing government and industry participated. In this study, 1404 test portions were analyzed of which 1152 were used in the statistical analysis. There were 448 positive by the VIDAS LMO2 assay and 457 positive by the standard culture methods. A chi2 analysis of each of the 5 food types, at the 3 inoculation levels tested, was performed. The resulting chi2 value, 0.36, indicates that overall, there are no statistical differences between the VIDAS LMO2 assay and the standard methods at the 5% level of significance.     

Evaluation of the VIDAS Listeria (LIS) immunoassay for the detection of Listeria in foods using demi-Fraser and Fraser enrichment broths, as modification of AOAC Official Method 999.06 (AOAC Official Method 2004.06)

A multilaboratory study was conducted to compare the VIDAS LIS immunoassay with the standard cultural methods for the detection of Listeria in foods using an enrichment modification of AOAC Official Method 999.06. The modified enrichment protocol was implemented to harmonize the VIDAS LIS assay with the VIDAS LMO2 assay. Five food types--brie cheese, vanilla ice cream, frozen green beans, frozen raw tilapia fish, and cooked roast beef--at 3 inoculation levels, were analyzed by each method. A total of 15 laboratories representing government and industry participated. In this study, 1206 test portions were tested, of which 1170 were used in the statistical analysis. There were 433 positive by the VIDAS LIS assay and 396 positive by the standard culture methods. A Chi-square analysis of each of the 5 food types, at the 3 inoculation levels tested, was performed. The resulting average Chi square analysis, 0.42, indicated that, overall, there are no statistical differences between the VIDAS LIS assay and the standard methods at the 5% level of significance.     

Evaluation of the BAX system for detection of Listeria monocytogenes in foods: collaborative study

A multilaboratory study was conducted to compare the automated BAX system and the standard cultural methods for detection of Listeria monocytogenes in foods. Six food types (frankfurters, soft cheese, smoked salmon, raw, ground beef, fresh radishes, and frozen peas) were analyzed by each method. For each food type, 3 inoculation levels were tested: high (average of 2 CFU/g), low (average of 0.2 CFU/g) and uninoculated controls. A total of 25 laboratories representing government and industry participated. Of the 2335 samples analyzed, 1109 were positive by the BAX system and 1115 were positive by the standard method. A Chi square analysis of each of the 6 food types, at the 3 inoculation levels tested, was performed. For all foods, except radishes, the BAX system performed as well as or better than the standard reference methods based on the Chi square results.     

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.     

Proficiency testing in food microbiology: experience from implementation of ISO/IEC 17043 and ISO/TS 22117

The proficiency testing (PT) scheme ??AQUA?? for food microbiology was organised by the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) according to ISO/IEC 17043 and ISO/TS 22117. This paper describes the IZSVe experience on the application of the above-mentioned standards for the PTs, with focus on the Enterobacteriaceae enumeration one. Freeze-dried food matrices contaminated with American Type Culture Collection bacterial strains were used as test samples for each microbiological PT organised by IZSVe. The sample homogeneity and stability were verified prior to distribution to participants and throughout the PT, respectively. The participating laboratories analysed samples using their routine methods, and results were transmitted to IZSVe. Data and methods used by each participating laboratory were analysed in order to evaluate the laboratory performance. With reference to the Enterobacteriaceae PT, the test samples were homogeneous and stable. In addition, most laboratory results were obtained using equivalent test methods. Statistical approaches applied to analyse data generated from all participating laboratories revealed similar outcomes as no significant outlying count and only 5?% of unacceptable results were observed. Finally, the z-score, with the standard deviation that does not vary from round toround, was applied to compare and to evaluate the performance of each laboratory over time highlighting possible persistent trends over several rounds.     

Enumeration of total aerobic microorganisms in foods by SimPlate Total Plate Count-Color Indicator methods and conventional culture methods: collaborative study

The relative efficacy of the SimPlate Total Plate Count-Color Indicator (TPC-CI) method (SimPlate 35 degrees C) was compared with the AOAC Official Method 966.23 (AOAC 35 degrees C) for enumeration of total aerobic microorganisms in foods. The SimPlate TPC-CI method, incubated at 30 degrees C (SimPlate 30 degrees C), was also compared with the International Organization for Standardization (ISO) 4833 method (ISO 30 degrees C). Six food types were analyzed: ground black pepper, flour, nut meats, frozen hamburger patties, frozen fruits, and fresh vegetables. All foods tested were naturally contaminated. Nineteen 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 among the SimPlate methods and their corresponding reference methods. Mean log counts between the 2 reference methods were also very similar. Repeatability (Sr) and reproducibility (SR) standard deviations were similar among the 3 method comparisons. The SimPlate method (35 degrees C) and the AOAC method were comparable for enumerating total aerobic microorganisms in foods. Similarly, the SimPlate method (30 degrees C) was comparable to the ISO method when samples were prepared and incubated according to the ISO method.     

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 a microwell DNA probe assay for detection of Listeria spp. in foods Performance-tested method 010403

A new DNA hybridization assay in microwell format for detection of Listeria spp. in foods and environmental samples was developed. This assay uses Listeria-specific oligonucleotide probes labeled with horseradish peroxidase and a photometrically determined end point. Validation studies with 15 different food commodities and a variety of environmental sample types were conducted to compare the performance of this alternative test versus reference methods. Meats, seafood, dairy products, and vegetables comprised the categories of food tested. Food samples were inoculated at 2 levels and refrigerated or frozen for at least 72 h. Uninoculated (negative) control samples were included in each trial. Samples were enriched according to the procedure recommended by either the U.S. Food and Drug Administration (FDA) or the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS). Samples enriched for 24 h were transferred to Oxford agar plates and incubated for 24 h. The surface of the plates was then swabbed and any growth present was transferred to phosphate buffer solution for the performance of the DNA assay. A standard confirmation procedure was used to compare the number of positive samples obtained with the DNA method versus reference methods. Statistical analyses of the results indicate that the proposed alternative method performs equally to cultural reference methods. The DNA assay is able to detect as low as 1 colony-forming unit of Listeria in a 25 g food sample, with results available as early as 48 h after the start of sample enrichment.     

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.     

Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes detection kit in combination with ShortPrep foodproof II Kit. Performance-Tested Method 070401

A method was developed for the detection of L. monocytogenes in food based on real-time polymerase chain reaction (PCR). This advanced PCR method was designed to reduce the time needed to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the Roche/BIOTECON Diagnostics ShortPrep foodproof II Kit (formerly called Listeria ShortPrep Kit) designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is performed by using the Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes Detection Kit. This kit provides primers and hybridization probes for sequence-specific detection, convenient premixed reagents, and different controls for reliable interpretation of results. For repeatability studies, 20 different foods, covering the 15 food groups recommended from the AOAC Research Institute (AOAC RI) for L. monocytogenes detection were analyzed: raw meats, fresh produce/vegetables, processed meats, seafood, egg and egg products, dairy (cultured/noncultured), spices, dry foods, fruit/juices, uncooked pasta, nuts, confectionery, pet food, food dyes and colorings, and miscellaneous. From each food 20, samples were inoculated with a low level (1-10 colony-forming units (CFU)/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, 5 uninoculated samples were prepared from each food. The food samples were examined with the test kits and in correlation with the cultural methods according to U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) or U.S. Department of Agriculture (USDA)/Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook. After 48 h of incubation, the PCR method in all cases showed equal or better results than the reference cultural FDA/BAM or USDA/FSIS methods. Fifteen out of 20 tested food types gave exactly the same amount of positive samples for both methods in both inoculation levels. For 5 out of 20 foodstuffs, the PCR method resulted in more positives than the reference method after 48 h of incubation. Following AOAC RI definition, these were false positives because they were not confirmed by the reference method (false-positive rate for low inoculated foodstuffs: 5.4%; for high inoculated foodstuffs: 7.1%). Without calculating these unconfirmed positives, the PCR method showed equal sensitivity results compared to the alternative method. With the unconfirmed PCR-positives included into the calculations, the alternative PCR method showed a higher sensitivity than the microbiological methods (low inoculation level: 100 vs 98.0%; sensitivity rate: 1; high inoculation level: 99.7 vs 97.7%; sensitivity rate, 1). All in-house and independently tested uninoculated food samples were negative for L. monocytogenes. The ruggedness testing of both ShortPrep foodproof II Kit and Roche/BIOTECON LightCycler foodproof L. monocytogenes Detection Kit showed no noteworthy influences to any variation of the parameters component concentration, apparatus comparison, tester comparison, and sample volumes. In total, 102 L. monocytogenes isolates (cultures and pure DNA) were tested and detected for the inclusivity study, including all isolates claimed by the AOAC RI. The exclusivity study included 60 non-L. monocytogenes bacteria. None of the tested isolates gave a false-positive result; specificity was 100%. Three different lots were tested in the lot-to-lot study. All 3 lots gave equal results. The stability study was subdivided into 3 parts: long-term study, stress test, and freeze-defrost test. Three lots were tested in 4 time intervals within a period of 13 months. They all gave comparable results for all test intervals. For the stress test, LightCycler L. monocytogenes detection mixes were stored at different temperatures and tested at different time points during 1 month. Stable results were produced at all storage temperatures. The freeze-defrost analysis showed no noteworthy aggravation of test results. The independent validation study examined by Campden and Chorleywood Food Research Association Group (CCFRA) demonstrated again that the LightCycler L. monocytogenes detection system shows a comparable sensitivity to reference methods. With both the LightCycler PCR and BAM methods, 19 out of 20 inoculated food samples were detected. The 24 h PCR results generated by the LightCycler system corresponded directly with the FDA/BAM culture results. However, the 48 h PCR results did not relate exactly to the FDA/BAM results, as one sample found to be positive by the 48 h PCR could not be culturally confirmed and another sample which was negative by the 48 h PCR was culturally positive.     

Advances in the thin layer chromatographic analysis of counterfeit pharmaceutical products: 2008–2019

Publications reporting thin layer chromatography (TLC) screening and high performance TLC (HPTLC)-densitometry quantification analyses of counterfeit pharmaceutical products are reviewed for the 2008–2019 period. Screening using TLC methods published in the Global Pharma Health Fund (GPHF) Minilab Manual and U.S. Food and Drug Administration (FDA) Compendium, as well as in other sources, are covered. Also included are publications on TLC analysis hyphenated with Raman and mass spectrometry; analyses of counterfeit traditional herbal medicines; earlier published reviews; transfer of screening methods for counterfeit pharmaceutical products in the Minilab Manual and FDA Compendium to HPTLC-densitometry using a model process; development of HPTLC-densitometry methods for pharmaceutical products not included in the Minilab Manual or FDA Compendium using the model process followed by development of corresponding Supplemental FDA Compendium TLC screening methods; and modified Minilab methods with simplified detection based on heating of silica gel F layers to produce fluorescence quenching zones (thermochemical activation) rather than detection using spray, dip, or vapor phase derivatization reagents. Some thoughts on future prospects for the field are also offered.     

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.     

Validation of the Hygicult E dipslides method in surface hygiene control: a Nordic collaborative study

A collaborative study with Enterobacteriaceae was conducted to validate Hygicult E dipslides by comparison with violet red bile glucose agar (VRBGA) contact plates and swabbing, using stainless steel surfaces artificially contaminated with microbes at various levels. Twelve laboratories participated in the validation procedure. The total number of collaborative samples was 108. The microbial level in each sample was assessed in triplicate by using the 3 above-mentioned methods. No Enterobacteriaceae were used at the low inoculation level. At the middle inoculation level, the percentages detached from the test surfaces were 16.6 with the Hygicult E method, 15.3 with the contact plate method, and 14.6 with swabbing; at the high innoculation level, the percentages were 14.5, 15.8, and 9.8, respectively. The percentage of acceptable results after the removal of outliers was 97.2. Repeatability relative standard deviations ranged from 33.4 to 44.9%; reproducibility relative standard deviations ranged from 45.2 to 77.1%. The Hygicult E dipslide, VRBGA contact plate, and swabbing methods gave similar results at all 3 microbial levels tested: <1.0 colony-forming units (CFU)/cm2 at the low level, 1.2-1.3 CFU/cm2 at the middle level (theoretical yield 8.0 CFU/cm2), and 1.2-2.0 CFU/cm2 at the high level (theoretical yield 12.5 CFU/cm2).     

VIDAS enzyme-linked immunoflourescent assay for detection of Listeria in foods: collaborative study

The VIDAS LIS method and the traditional culture methods for detection of Listeria species in food were evaluated in a multilaboratory comparative study. The 6 foods tested were either naturally contaminated or inoculated with 3 different concentrations of Listeria. Results for each food and each contamination level with the VIDAS LIS method were as good as or better than those obtained with the traditional culture method. Of 1558 samples tested, 935 were positive: 839 by the VIDAS method and 809 by standard culture methods. Overall false negative rates were 10.3 and 13.5% for the VIDAS LIS and culture methods, respectively. The false positive rate for the VIDAS LIS assay was 1.4% based on 9 VIDAS LIS positive assays that did not confirm positive by isolation of Listeria. The agreement between the VIDAS LIS and culture methods for all samples tested was 86%.