Validation of the CompactDry "Nissui" YMR for Enumeration of Yeasts and Molds in a Variety of Foods: AOAC Performance Tested MethodSM 092002.

BACKGROUND: The CompactDry "Nissui" YMR is a ready-to-use dry media sheet using a chromogenic medium with selective agents for the detection and enumeration of yeasts and molds in products after incubation at 25 ± 1°C for 3 days. OBJECTIVE: The CompactDry "Nissui" YMR method was validated in order to achieve AOAC Performance Tested MethodsSM certification. METHOD: The performance of the CompactDry "Nissui" YMR was compared to that of ISO 21527-1:2008 for 10 matrixes including cooked prawns, deli vegetable salad, tuna pâté, fermented yogurt drink, spinach and ricotta quiche, egg custard tarts, fruit and vegetable smoothie, cream cheese, egg salad sandwich, and deli pasta salad. Performance indicators included repeatability, difference of means (DOM), and inclusivity/exclusivity. RESULTS: After log10 transformation of the data, the relative standard deviation of repeatability (RSDr) was <10% for all 30 materials (10 matrixes each at 3 levels) analyzed by the CompactDry "Nissui" YMR method and for 29 of the 30 matrix/level combinations analyzed by the reference method. The DOM ranged from -0.284 (-0.310, -0.257) log10 CFU/g to 0.307 (-0.013, 0.627) log10 CFU/g. Method equivalence was demonstrated in 29 of the 30 matrix/level combinations based on the 90% confidence interval of the DOM being within (-0.5, 0.5). All 51 inclusivity strains showed expected or atypical results, and all 32 exclusivity organisms showed no growth on the medium. The method was shown to be robust to changes in sample volume, incubation temperature, and incubation time, and data are presented supporting product consistency and a 24-month shelf life. CONCLUSIONS: The CompactDry "Nissui" YMR method is validated for the determination of yeasts and molds in a variety of matrixes. HIGHLIGHTS: The CompactDry "Nissui" YMR method is equivalent to the ISO 21527-1:2008 reference method and is suitable for Performance Tested MethodsSM certification for the matrixes tested.

CompactDry "Nissui" ETC for Enumeration of Enterococci in a Variety of Foods: AOAC Performance Tested MethodSM 111902.

BACKGROUND: The CompactDry "Nissui" ETC is a ready-to-use dry media sheet using a chromogenic medium with selective agents for the detection and enumeration of Enterococcus spp. After a 20-24 h incubation at 37 ± 1°C, Enterococcus colonies appear blue. OBJECTIVE: The performance of the CompactDry "Nissui" ETC was compared to that of the Nordic Committee on Food Analysis (NMKL) reference method 68, Enterococcus, Determination in Foods and Feeds, for 10 matrixes including cream, custard, lettuce, parsley, pasta salad, frozen ground beef patties, fresh cooked prawns, sandwiches, raw beef, and tuna pate. METHODS: Performance indicators included repeatability, difference of means (DOM), robustness and inclusivity/exclusivity. RESULTS: For all 30 materials (10 matrixes each inoculated at three levels), the relative standard deviation of repeatability (RSDr) was <10% for both methods. Most materials yielded RSDr <5%, with five materials between 5 and 10% for the CompactDry "Nissui" ETC method and ten materials between 5 and 10% for the NMKL 68 method. The DOM ranged from -0.240 (95% CI-0.363, -0.118) to 0.228 (95% CI 0.041, 0.415) log10 CFU/g, well within the -0.5 to 0.5 log10 CFU/g range generally accepted as microbiologically insignificant. Both the reference method and this method have limitations of the species detected. CONCLUSIONS: The CompactDry "Nissui" ETC was validated for the enumeration of Enterococcus species in cream, custard, lettuce, parsley, pasta salad, frozen ground beef patties, fresh cooked prawns, sandwiches, raw beef, and tuna pate.

Matrix Extension Study: Validation of the Compact Dry CF Method for Enumeration of Total Coliform Bacteria in Selected Foods.

The Compact Dry "Nissui" CF method, Performance Tested Method(SM) 110401, was originally certified for enumeration of coliform bacteria by the AOAC Research Institute Performance Tested Methods(SM) program for raw meat products. Compact Dry CF is a ready-to-use dry media sheet, containing a cold-soluble gelling agent, a chromogenic medium, and selective agents, which are rehydrated by adding 1 mL of diluted sample. Coliform bacteria produce blue/blue-green colonies on the Compact Dry CF, allowing a coliform colony count to be determined in the sample after 24 ± 2 h incubation. A validation study was organized by Campden BRI (formerly Campden and Chorleywood Food Research Association Technology, Ltd), Chipping Campden, United Kingdom, to extend the method's claim to include cooked chicken, fresh bagged prewashed shredded iceberg lettuce, frozen fish, milk powder, and pasteurized 2% milk. Campden BRI collected single-laboratory data for cooked chicken, lettuce, frozen fish, and milk powder, whereas a multilaboratory study was conducted on pasteurized milk. Thirteen laboratories participated in the interlaboratory study. The Compact Dry CF method was compared to ISO 4832:2006 "Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coliforms-Colony-count technique," the current version at the time this study was conducted. Each matrix was evaluated at either four or five contamination levels of coliform bacteria (including an uncontaminated level). After logarithmic transformation of counts at each level, the data for pasteurized whole milk were analyzed for sr, sR, RSDr, and RSDR. Regression analysis was also performed and r(2) was reported. Mean difference between methods with 95% confidence interval (CI) was calculated. A log10 range of -0.5 to 0.5 for the CI was used as the acceptance criterion to establish significant statistical difference between methods. In the single-laboratory evaluation (for cooked chicken, lettuce, frozen fish, and milk powder), sr and RSDr values were analyzed and r(2) was reported. Statistical differences were indicated between the Compact Dry CF and ISO 4832 methods in two of five contamination levels tested for lettuce, and in the low contamination levels for cooked chicken, frozen fish, and dry milk powder. For the low levels of cooked chicken, frozen fish, and milk powder, only a few colonies were recovered for each method, and thus not a true indication of the methods' performance. In most cases, mean differences between the Compact Dry CF and ISO 4832 methods were small (<0.5 log10), with CIs within the acceptance criterion. The sr and RSDr values were similar for both methods, and r(2) values were >0.94 for all matrixes. In the multilaboratory study, no statistical differences were indicated between methods. The sr, RSDr, sR, and RSDr values were similar for each method and even slightly smaller for the Compact Dry CF. The r(2) value was 0.99. The Compact Dry CF method offers comparable results to ISO 4832 in a space saving, easy-to-use format.

Matrix Extension Study: Validation of the Compact Dry EC Method for Enumeration of Escherichia coli and non-E. coli Coliform Bacteria in Selected Foods.

The Compact Dry "Nissui" EC method, originally certified by the AOAC Research Institute Performance Test Method(SM) program for enumeration of Escherichia coli and non-E. coli coliforms in raw meat products (Performance Tested Method(SM) 110402), has undergone an evaluation to extend the method's claim to cooked chicken, prewashed bagged shredded iceberg lettuce, frozen cod filets, instant nonfat dry milk powder, and pasteurized milk (2% fat). Compact Dry EC is a ready-to-use dry media sheet containing a cold-soluble gelling agent, selective agents, and a chromogenic medium, which are rehydrated by adding 1 mL diluted sample. E. coli form blue/blue-purple colonies, whereas other coliform bacteria form red/pink colonies. Users can obtain an E. coli count (blue/blue-purple colonies only) and a total coliform count (red/pink plus blue/blue-purple colonies) after 24 ± 2 h of incubation at 37 ± 1°C. The matrix extension study was organized by Campden BRI (formerly Campden and Chorleywood Food Research Association Technology, Ltd), Chipping Campden, United Kingdom. Method comparison data for cooked chicken, prewashed bagged shredded iceberg lettuce, frozen cod filets, and instant nonfat dry milk powder were collected in a single-laboratory evaluation by Campden BRI. A multilaboratory study was conducted on pasteurized milk (2% fat), with 13 laboratories participating. The Compact Dry EC method was compared to ISO 16649-2:2001 "Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli-Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide" and to ISO 4832:2006 "Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coliforms-Colony-count technique," the current standards at the time of this study. Each matrix was evaluated separately for E. coli and non-E. coli coliforms at each contamination level (including an uncontaminated level). In the single-laboratory evaluation (cooked chicken, prewashed bagged shredded iceberg lettuce, frozen cod filets, and instant nonfat dry milk powder), colony counts were logarithmically transformed, and then the data were analyzed at each level for sr, RSDr, and mean difference between methods with 95% confidence intervals (CIs). A CI outside a range of -0.5 to 0.5 on the log10 mean difference between methods was used as the criterion to establish a significant statistical difference. In the multilaboratory study on pasteurized milk, after logarithmic transformation, the data were analyzed for sR and RSDR in addition to sr, RSDr, and mean difference with 95% CIs. Regression analysis was performed on all matrixes and reported as r(2). In the single-laboratory evaluation, statistical differences were indicated between the Compact Dry EC and ISO 16649-2 methods for the enumeration of E. coli in two of five contamination levels tested for lettuce, and in the low contamination level for cooked chicken. For the cooked chicken and lettuce at the low level, only a few colonies were recovered for each method, and thus not a true indication of the methods' performance. For the high contamination level of lettuce, counts varied within the sets of five replicates more than 10-fold for each method, which may have contributed to the significant difference. Statistical differences were also indicated between the Compact Dry EC and ISO 4832 methods for the enumeration of coliforms in two of five contamination levels tested for lettuce, two of five contamination levels of milk powder, and in the low contamination level for frozen fish. For the lowest levels of frozen fish and milk powder, only a few colonies were recovered for each method. For the lettuce and the other level of milk powder, counts varied within the sets of five replicates more than 10-fold for each method, which may have contributed to the significant differences indicated in the those contamination levels. In most cases, mean differences between the Compact Dry EC and International Organization of Standardization (ISO) methods were well below 0.5 log10, and the CIs were within the acceptance criterion (-0.5 to 0.5). The sr and RSDr values were similar for both methods, and r(2) values were >0.92 for all comparisons. In the multilaboratory study, no statistical differences were indicated between the methods. The sr, RSDr, sR, and RSDr values were similar for each method and even slightly smaller in most cases for the Compact Dry EC. The r(2) value was 0.97 in comparison to ISO 16649-2, and 0.99 in comparison to ISO 4832. The Compact Dry EC offers comparable results to the ISO standard plating methods in a space saving, easy-to-use format.

Matrix Extension Study: Validation of the Compact Dry TC Method for Enumeration of Total Aerobic Bacteria in Selected Foods.

A validation study was conducted to extend the matrix claim for the Nissui Compact Dry Total Count (TC), Performance Tested Method(s)(SM) (PTM) Certification No. 010404, to cooked chicken, lettuce, frozen fish, milk powder, and pasteurized whole milk. The method was originally certified by the AOAC Research Institute Performance Tested Method(s)(SM) Program for raw meat products. The Compact Dry TC is a ready-to-use dry media sheet that is rehydrated by adding 1 mL of diluted sample. A total aerobic colony count can be determined in the sample following 48 h of incubation. Matrix extension studies were conducted by Campden BRI (formerly Campden and Chorleywood Food Research Association Technology Limited), Chipping Campden, UK. Single-laboratory data were collected for cooked chicken, lettuce, frozen fish, and milk powder, whereas a multilaboratory study was conducted on pasteurized milk. Fourteen laboratories participated in the collaborative study. The Compact Dry TC was tested at two time points, 48 ± 3 h and 72 ± 3 h and compared with the current International Organization for Standardization (ISO) method at the time of the study, ISO 4833:2003 (this standard is withdrawn and has been replaced by: ISO 4833-1:2013 and ISO 4833-2:2013), Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of microorganisms-Colony-count technique at 30°C. The data were logarithmically transformed and evaluated for repeatability (plus reproducibility for pasteurized milk), RSD of repeatability (plus RSD of reproducibility for milk), r(2), and mean difference between methods with 95% confidence interval (CI). A CI outside of (-0.5 to 0.5) on the log10 mean difference was used as the criterion to establish significant statistical difference between methods. No significant differences were found between the Compact Dry TC 48 and 72 h time points, with the exception of one contamination level of cooked chicken and one contamination level of dry milk powder. Mean differences were small at these levels (<0.5 log10), but the upper CIs were above 0.5. Statistical differences were indicated between the Compact Dry TC and ISO 4833 in two of five contamination levels tested each for lettuce and frozen fish. In each case, mean differences were >0.5 log10, and the total aerobic colony count was higher for the ISO method. In most cases, mean differences between the Compact Dry and ISO methods were small (<0.5 log10) with CIs within the acceptance criterion. Repeatability, reproducibility, and RSD were similar for both methods, and r(2) values were >0.97 for all matrixes. The Compact Dry TC, at 48 h, offers the advantage of a shorter time to results than ISO 4833 in an easy-to-use format.

Evaluation of the dry sheet medium (Compact Dry ETB(R)) for enumerating Enterobacteriaceae in meat samples.

Compact Dry ETB(R) (CD-ETB, ETB; Enterobacteriaceae) , a ready-to-use and self-diffusible dry medium sheet culture system for the detection and enumeration of Enterobacteriaceae, was evaluated. A total of 35 Enterobacteriaceae strains, which were studied for the inclusivity study, grew and formed red-purple colored colonies on the CD-ETB(R). When 17 gram-negative bacteria other than Enterobacteriaceae were inoculated for the exclusivity study, 2 strains grew and formed red-purple colonies, 9 strains formed colorless colonies, and 6 strains failed to grow. A total of 43 gram-positive bacteria and 3 yeasts failed to grow. The CD-ETB method was compared with the Violet Red Bile Glucose (VRBG) agar method according to ISO 21528-2 and the 3M Petrifilm(TM) EB (3M-EB(R)) method in 53 meat samples. The correlation coefficients between CD-ETB(R) and VRBG agar, CD-ETB(R) and 3M-EB(R), and 3M-EB(R) and VRBG agar were 0.962, 0.992 and 0.960, respectively.

Evaluation of the compact dry X-SA method for enumerating Staphylococcus aureus in artificially contaminated food samples.

Compact Dry X-SA (CD-XSA), a ready-to-use and self-diffusible dry medium sheet culture system for the detection and enumeration of Staphylococcus aureus, was evaluated. A total of 50 S. aureus strains, which were studied for the inclusivity study, grew as blue-colored colonies on the CD-XSA. When 114 bacteria other than S. aureus and 3 yeasts were inoculated for the exclusivity study, 37 strains produced white colonies, and 4 strains produced blue colonies, and 3 strains produced magenta colonies, while 73 other strains failed to grow. The CD-XSA method was compared with the mannitol salt agar with egg yolk (MSEY) method, the Baird-Parker agar (BP) method and the 3M Petrifilm(TM) STX (3M-STX) method in 105 artificially contaminated food samples. The correlation coefficients between CD-XSA and MSEY, CD-XSA and BP, and CD-XSA and 3M-STX were 0.945, 0.960 and 0.977, respectively.

Evaluation of the Compact Dry VP method for screening raw seafood for total Vibrio parahaemolyticus.

Compact Dry VP (CDVP) is a ready-to-use method for enumerating Vibrio parahaemolyticus in food. The presterilized plates contain a culture medium comprising peptone, NaCl, bile salts, antibiotics, chromogenic substrates, and polysaccharide gum as a cold water-soluble gelling. After diluting raw seafood samples in a phosphate-buffered saline solution, a 1-ml aliquot was inoculated onto the center of the plate and allowed to diffuse by capillary action. Blue-green colonies forming on the plates were counted after 18 to 20 h of incubation at 35 degrees C. A total of 85 V. parahaemolyticus strains (62 tdh+ strains and 23 tdh- strains) were studied for inclusivity, 81 (95.3 %) of which produced blue-green colonies. When 97 strains (14 strains of Vibrio spp., 33 strains of coliform bacteria, and 50 strains of noncoliform bacteria) were assessed for exclusivity, 10 strains of Vibrio spp. produced non-blue-green colonies, and 87 strains failed to grow. The CDVP and U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM) methods were compared with the use of four different types of raw seafood that were inoculated with four different V. parahaemolyticus strains. For raw tuna and oysters, the FDA-BAM colony lift method was used, whereas the FDA-BAM most-probable-number method was used for salmon and scallop. The linear correlation coefficients between the CDVP and FDA-BAM methods were 0.99 for fresh raw tuna, 0.95 for fresh raw oysters, 0.95 for frozen raw salmon, and 0.95 for frozen raw scallops. These results suggest that the CDVP method is useful for screening raw seafood for V. parahaemolyticus.

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."

Comparison of the compact dry CF with the most probable number method (AOAC official method 966.24) for enumeration of coliform bacteria in raw meats. Performance-Tested Methods" 110401.

Compact Dry CF is a ready-to-use test method for the enumeration of 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 into 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 CF medium plates were validated with 5 different raw meats. The performance tests were conducted at 35 degrees C. In all studies performed, no apparent differences were observed between the Compact Dry CF method and the AOAC Official Method 966.24 results. For the accuracy claim (n = 75), a correlation factor of r2 = 0.91 (coliform) could be assigned, as stated in the application for Performance-Tested Method. No significant variations in coliform bacterial counts were observed with different production lots or plates of diverse storage age by the quality consistency and storage robustness studies.

Comparison of the compact dry YM with the FDA BAM method for enumeration of yeasts and molds in fruit-based products. Performance-Tested Method 100401.

Compact Dry YM is a ready-to-use test method for the enumeration of yeasts and molds in fruit-based products. The plates are presterilized and contain nonwoven incorporated nutrients supplemented with antibiotics and a chromogenic enzyme substrate, 5-bromo-4-chloro-3-indoxyl phosphate, p-toluidine salt (X-phos), to facilitate counting dyes, and a cold water-soluble gelling agent. The medium should be rehydrated with 1 mL of diluted sample inoculated onto the center of the self-diffusible medium, allowing the solution to diffuse immediately by capillary action. The plate can be incubated at 25 degrees C for 7 days. The Compact Dry YM medium plates were validated as an analysis tool determining colony forming units of yeasts and molds from a variety of fruit-based products using 5 different fruit products. The performance tests were conducted at 25 degrees C. In all studies performed, no apparent differences were observed between the Compact Dry YM method and the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA BAM) method results (P > 0.05). For the accuracy claim (n = 75), a correlation factor of r2 = 0.99 could be assigned, as stated in the application for Performance-Tested Method.

Comparison of the compact dry TC method with the standard pour plate method (AOAC official method 966.23) for determining aerobic colony counts in food samples: Performance-tested method.

Compact Dry TC qualifies as a rapid method kit for determining aerobic colony counts in foods. The plates are presterilized and contain culture medium and a cold-soluble gelling agent. The medium is rehydrated by inoculating 1 mL diluted sample into the center of the self-diffusible medium and allowing the solution to diffuse by capillary action. The plates can then be incubated and the colonies counted without any additional steps. The Compact Dry TC method was validated with 5 different raw meats. The performance tests were conducted at 35 degrees and 30 degrees C. In all required performance studies, no apparent differences were observed between the Compact Dry TC method and the Standard Pour Plate method (AOAC Official Method 966.23) for the detection level of aerobic microorganisms. For the accuracy claim (n = 60), a correlation factor of r2(35) = 0.9977 (35 degrees C) and r2(30) = 0.9932 (30 degrees C) could be assigned, as stated in the application for "Performance Tested Method." Quality consistency and storage robustness studies, showed no significant variations in plate count results with different production lots or plates of diverse storage age.