Bacteriological Analytical Manual Reference Research Articles

Comparison of Different Culture Methods for the Detection of Bacillus cereus Group in Cosmetics.

The U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) reference culture method uses Modified Letheen Broth (MLB) for microbiological analyses for all types of cosmetic products. This study evaluated the effectiveness of MLB and Tryptone Azolectin Tween (TAT) broths using BAM reference culture method for cosmetics. Pure spore suspensions of B. cereus group members were experimentally spiked (McF: 0.5) into cosmetic products. After an aging period of 72 h, the products were analyzed using MLB and TAT broth. The enumeration of the cells was performed on B. cereus group selective plates Bacillus cereus rapid agar (BACARA) and Mannitol Yolk Polymyxin (MYP) plates. No statistical difference (p > 0.05) was found for the recovery of cells from the liquid products using either medium (MLB or TAT broth) and the selective plates. In solid/powder products, a combination of Tween 80 and MLB detected significantly more cells (p < 0.05) than combination of Tween 80 and TAT broth. The microbial counts on BACARA showed no significant differences (p > 0.05). However, when assessing cream/oil-based products, the number of cells detected by use of Tween 80/TAT broth was significantly higher than Tween 80/MLB, and MYP showed significantly higher counts than BACARA. This study showed that relative effectiveness of MLB vs. TAT for recovering of B. cereus group cells varied depending on the variety of formulation, and combination of preservatives of the tested cosmetic products. The findings suggest additional studies are needed to explore recovery of other relevant microorganisms that may contaminate cream/oil-based cosmetics.

Matrix Extension Study: Listeria Right Now™ Test for Detection of Listeria spp. from Selected Environmental Surfaces Without Enrichment: AOAC Performance Tested MethodSM 081802.

Listeria Right Now™ is a novel, enrichment-free test for the detection of Listeria spp. in swab samples taken from environmental surfaces. Results are available in less than 1 h. In a previous Performance Tested MethodSM (PTM) study, the test was validated for swab samples from stainless-steel and sealed concrete surfaces. A PTM matrix extension study was conducted to validate the method for the detection of Listeria spp. in swab samples from ceramic tile, plastic, and rubber surfaces. Performance of the Listeria Right Now method was compared to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedure for the detection of Listeria spp. in swab samples taken from inoculated ceramic tile, plastic, and rubber surfaces. Data were analyzed using a probability of detection model. There were no significant differences in performance between the Listeria Right Now and reference culture methods for any of the three surfaces tested, as determined by probability of detection analysis. The Listeria Right Now method is an effective procedure for the detection of Listeria spp. from a variety of environmental surfaces. Listeria Right Now provides accurate results, without enrichment, in real time. This enables food industry personnel to react swiftly to suspected Listeria contamination incidents.

Validation of the Listeria Right NowTM Test for Detection of Listeria spp. from Selected Environmental Surfaces Without Enrichment.

Background: Listeria Right NowTM is a novel, enrichment-free molecular method for detection of Listeria spp. in swab samples from environmental surfaces. The test provides results in real time, indicating the current or recent presence of Listeria spp. in the environment. After sampling, the entire contents of the swab are subject to sample processing, releasing large quantities of target ribosomal RNA molecules into the lysate. A portion of the lysate is then tested using the ANSR® for Listeria isothermal nucleic acid amplification assay. Objective: A Performance Tested MethodSM study was conducted to validate the method for detection of Listeria spp. in swab samples from stainless steel and sealed concrete surfaces. Methods and Results: In inclusivity testing, 60 of 60 Listeria spp. strains tested positive. In exclusivity testing, 31 of 31 nontarget bacterial strains tested negative. In LOD testing, the test was able to detect as few as 2 CFU of L. monocytogenes applied to a stainless steel surface. In matrix testing of inoculated stainless steel and sealed concrete surfaces, there were no statistically significant differences in method performance comparing the Listeria Right Now and U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedures as determined by probability of detection analysis. In robustness testing, modest changes to three assay operating parameters simultaneously did not significantly affect performance of the test. Conclusions and Highlights: Results can be obtained in less than 1 h using the Listeria Right Now test, allowing food industry personnel to take immediate corrective action in the case of Listeria contamination incidents.

Matrix Extension Study: Listeria Right Now™ Test for Detection of Listeria spp. from Selected Environmental Surfaces Without Enrichment

Background: Listeria Right Now™ is a novel, enrichment-free test for the detection of Listeria spp. in swab samples taken from environmental surfaces. Results are available in less than 1 h. In a previous Performance Tested MethodSM (PTM) study, the test was validated for swab samples from stainless-steel and sealed concrete surfaces. Objective: A PTM matrix extension study was conducted to validate the method for the detection of Listeria spp. in swab samples from ceramic tile, plastic, and rubber surfaces. Methods: Performance of the Listeria Right Now method was compared to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedure for the detection of Listeria spp. in swab samples taken from inoculated ceramic tile, plastic, and rubber surfaces. Data were analyzed using a probability of detection model. Results: There were no significant differences in performance between the Listeria Right Now and reference culture methods for any of the three surfaces tested, as determined by probability of detection analysis. Conclusions: The Listeria Right Now method is an effective procedure for the detection of Listeria spp. from a variety of environmental surfaces. Highlights: Listeria Right Now provides accurate results, without enrichment, in real time. This enables food industry personnel to react swiftly to suspected Listeria contamination incidents.

Validation of a Modified ANSR® for E. coli O157:H7 Method for Detection of E. coli O157:H7 in Select Foods.

Background: The ANSR method is based on isothermal nucleic acid amplification technology. The modifications to assay components improve sensitivity of the assay and robustness of the internal positive control. Objective: A Performance Tested MethodSM validation study was conducted to assess performance of a modified version of the ANSR® for Escherichia coli O157:H7 method. Methods: The validation study included inclusivity/exclusivity, matrix, robustness, accelerated stability, and independent laboratory testing. Results: In inclusivity testing of 55 strains of E. coli O157:H7 and E. coli O157:NM variants, all strains produced positive results. In exclusivity testing of 41 strains including E. coli of other serotypes and bacteria of closely related genera, all strains produced negative results. In matrix testing of beef trim, raw ground beef, spinach, and sprout-irrigation water, ANSR method performance was compared with that of the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook or the U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedures. Conclusions: all trials, ANSR method performance was not statistically different from that of the reference methods. Results of independent laboratory testing of ground beef corroborated those of internal testing. Introducing modest changes to three assay operating parameters did not materially affect ANSR method performance. Finally, accelerated stability testing results of three independently manufactured lots of ANSR reagents support a shelf-life of 1 year when stored at 2-8°C.

Validation of the ANSR(®) E. coli O157:H7 Method for Detection of E. coli O157:H7.

A performance validation of the ANSR(®) for E. coli O157:H7 method was conducted in selected food matrixes. This assay uses selective nicking enzyme amplification technology to amplify target genes. Samples are enriched for 12-24 h and then lysed. The assay is completed within 40 min using real-time detection in a combination incubator/fluorescence detector and software. When 44 distinct strains of Escherichia coli O157:H7 and 6 strains of E. coli O157:NM were tested for inclusivity, all 50 strains produced positive results. In exclusivity testing, 57 strains representing 33 species of closely related Gram-negative bacteria belonging to the Enterobacteriaceae family, including 11 non-H7 O157 strains and shiga toxin-producing E. coli other than O157:H7, were evaluated. All 57 nontarget strains generated negative ANSR assay results. Using 80% lean ground beef and beef trim (approximately 20% fat), ANSR method performance was compared to the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedure. ANSR performance with baby spinach and sprout irrigation water was measured against the U.S. Food and Drug Administration Bacteriological Analytical Manual reference method. ANSR method performance was not statistically different to that of the reference methods using two different enrichment options. For ground beef and beef trim, the standard enrichment in modified Tryptone Soya Broth can be analyzed using the ANSR assay with a 1:10 dilution of the enrichment in phosphate-buffered saline and produces equivalent results to the reference method. Additionally, in most matrixes tested (exception is spinach which required 24 h enrichment) the assay offers great efficiency and flexibility over the reference method with a 12-24 h single-step enrichment. Equivalent results were observed at both time points (12 and 24 h) to reference methods. Small changes to the assay parameters minimally affected ANSR method performance. Finally, accelerated stability results from three independently manufactured lots support a shelf-life of 6 months when stored at 4°C.

Validation of the ANSR(®) Listeria monocytogenes Method for Detection of Listeria monocytogenes in Selected Food and Environmental Samples.

Work was conducted to validate performance of the ANSR(®) for Listeria monocytogenes method in selected food and environmental matrixes. This DNA-based assay involves amplification of nucleic acid via an isothermal reaction based on nicking enzyme amplification technology. Following single-step sample enrichment for 16-24 h for most matrixes, the assay is completed in 40 min using only simple instrumentation. When 50 distinct strains of L. monocytogenes were tested for inclusivity, 48 produced positive results, the exceptions being two strains confirmed by PCR to lack the assay target gene. Forty-seven nontarget strains (30 species), including multiple non-monocytogenes Listeria species as well as non-Listeria, Gram-positive bacteria, were tested, and all generated negative ANSR assay results. Performance of the ANSR method was compared with that of the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedure for detection of L. monocytogenes in hot dogs, pasteurized liquid egg, and sponge samples taken from an inoculated stainless steel surface. In addition, ANSR performance was measured against the U.S. Food and Drug Administration Bacteriological Analytical Manual reference method for detection of L. monocytogenes in Mexican-style cheese, cantaloupe, sprout irrigation water, and guacamole. With the single exception of pasteurized liquid egg at 16 h, ANSR method performance as quantified by the number of positives obtained was not statistically different from that of the reference methods. Robustness trials demonstrated that deliberate introduction of small deviations to the normal assay parameters did not affect ANSR method performance. Results of accelerated stability testing conducted using two manufactured lots of reagents predicts stability at the specified storage temperature of 4°C of more than 1 year.

Validation of the NeoFilm for Yeast and Mold Method for Enumeration of Yeasts and Molds in Select Foods.

NeoFilm Yeast and Mold (Y&M), also known as Sanita-kun Yeasts and Molds, is a simple, effective device used for the enumeration of yeasts and molds. It consists of a nonwoven fabric on which a layer of microbial nutrients is deposited in a film. A 1 mL sample homogenate is applied to the membrane and this, in turn, is incubated for 48-72 h at 25°C. Sample homogenates were prepared using two different diluents for customer convenience: phosphate buffered saline (PBS) and 0.1% peptone water. In comparative testing of breaded chicken nuggets, dry pet food, orange juice concentrate, yogurt, and cake mix, there were statistically significant differences in the counts obtained by the NeoFilm Y&M and U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture methods only in the following instances: medium level for orange juice with PBS as diluent and low level for pet food with 0.1% peptone water as diluent, where reference method counts were higher than those of NeoFilm; medium level for cake mix with PBS, and low and medium levels for cake mix with 0.1% peptone water, where NeoFilm produced higher counts than the reference method. In addition to the method comparison study with five matrixes, robustness and stability/lot-to-lot testing were also performed. Results of robustness testing showed no significant effect on results even with perturbation to three assay parameters simultaneously. Results of testing of three lots of devices ranging in age from 2 to 26 months post-manufacture showed no significant differences in performance.

Immunomagnetic separation and visual fluorescence detection of Salmonella spp., using AOAC approved SAS Molecular Tests.

The SAS Molecular Tests method for detection of Salmonella spp. in various food matrixes has been certified by the AOAC Research Institute and designated Performance Tested Method No. 021202. The current method modification includes the optional immunomagnetic separation (IMS) to enrich the bacteria as well as optional visual fluorescence readout without the use of a turbidimeter. The modifications were validated against the U.S. Department of Agriculture/ Food Safety and Inspection Service Microbiology Laboratory Guidebook (MLG) and the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) reference methods. Food matrixes (chicken carcass rinse, beef trim, and spinach) were inoculated with low levels of Salmonella spp. (0.2-2 CFU/test portion) to generate fractional positives (5-15) in 20 inoculated samples. Samples were enriched with SAS Enrichment medium and incubated at 42 +/- 1 degree C. Enrichments were tested directly and subjected to anti-Salmonella IMS prior to the SAS Molecular Tests. Results were determined via visual fluorescence and via turbidity using a turbidimeter. All replicates were confirmed using the MLG or BAM reference method procedures, regardless of presumptive result. The SAS Molecular Tests Salmonella Detection modified methods were determined to be equivalent to the reference methods for the detection of Salmonella in chicken carcass rinse, beef trim, and fresh spinach. The inclusion of IMS in the modified method improved the detection rate of Salmonella in chicken carcass rinses and spinach. The optional use of visual fluorescent reagent and heat block either with IMS or without IMS produced results that were comparable to the results obtained from using a real-time turbidimeter.

SAS molecular tests Salmonella detection kit. Performance tested method 021202.

The SAS Molecular tests Salmonella Detection method, a Loop-mediated Isothermal Amplification method, performed as well as or better than the U.S. Department of Agriculture-Food Safety Inspection Service Microbiology Laboratory Guidebook and the U.S. Food and Drug Administration Bacteriological Analytical Manual reference methods for ground beef, beef trim, ground turkey, chicken carcass rinses, bagged mixed lettuce, and fresh spinach. The ground beef (30% fat, 25 g test portion), poultry matrixes and leafy greens were validated in a 6-7 h enrichment, and ground beef (30% fat, 375 g composite test portion) and beef trim (375 g composite test portion) were validated in a 16-20 h enrichment. The method performance for meat and leafy green matrixes was shown to be acceptable under conditions of co-enrichment with Escherichia coli 0157. Thus, after a short 6-7 h co-enrichment step, ground beef, beef trim, lettuce, and spinach can be tested for both Salmonella and E. coli O157. Inclusivity and exclusivity testing revealed no false negatives and no false positives among the 100 Salmonella serovars and 30 non-Salmonella species examined. The method was shown to be robust when enrichment time, DNA extract hold time, and DNA volume were varied.

SAS molecular tests Escherichia coli O157 detection kit. Performance tested method 031203.

The SAS Molecular tests Escherichia coli O157 Detection method, a loop-mediated isothermal amplification method, performed as well as or better than the U.S. Department of Agriculture, Food Safety Inspection Service Microbiology Laboratory Guidebook and the U.S. Food and Drug Administration Bacteriological Analytical Manual reference methods for ground beef, beef trim, bagged mixed lettuce, and fresh spinach. Ground beef (30% fat, 25 g test portion) was validated for 7-8 h enrichment, leafy greens were validated in a 6-7 h enrichment, and ground beef (30% fat, 375 g composite test portion) and beef trim (375 g composite test portion) were validated in a 16-20 h enrichment. The method performance for meat and leafy green matrixes was also shown to be acceptable under conditions of co-enrichment with Salmonella. Thus, after a short co-enrichment step, ground beef, beef trim, lettuce, and spinach can be tested for both Salmonella and E. coli O157. The SAS Molecular tests Salmonella Detection Kit was validated using the same test portions as for the SAS Molecular tests E. coli O157 Detection Kit and those results are presented in a separate report. Inclusivity and exclusivity testing revealed no false negatives and no false positives among the 50 E. coli 0157 strains, including H7 and non-motile strains, and 30 non-E. coli O157 strains examined. Finally, the method was shown to be robust when variations to DNA extract hold time and DNA volume were varied. The method comparison and robustness data suggest a full 7 h enrichment time should be used for 25 g ground beef test portions.

RAPID' Salmonella Chromogenic Medium

RAPID'Salmonella is a chromogenic medium for isolation and detection of Salmonella spp. in food, based on two enzymatic activities. All presumptive Salmonella-positive colonies are magenta, including lactose-positive Salmonella. S. Typhi, and S. Paratyphi serotypes, due to detection of C8 esterase activity. In order to differentiate Salmonella from other Enterobacteriaceae, the medium includes a second chromogenic substrate. As part of an Emergency Response Validation due to a massive outbreak and subsequent recall, peanut butter was tested to compare the performance of RAPID'Salmonella to the U.S. Food and Drug Administration's Bacteriological Analytical Manual reference method for detection of Salmonella. Sensitivity and specificity for RAPID'Salmonella were 100%.

1-2 Test

A study was conducted to determine the efficacy of detecting low contamination levels of Salmonella in peanut butter using the 1-2 Test. This study was conducted under the AOAC Research Institute Emergency Response Validation program. A set of samples was analyzed by the 1-2 Test and the U.S. Food and Drug Administration's Bacteriological Analytical Manual reference method. Among the 90 total samples and controls, 32 samples were positive by the 1-2 Test and the reference method. Statistical analyses of these paired samples indicated that for all levels analyzed, the difference between the 1-2 Test and reference method results were not statistically different at the 5% level.

Assurance GDS for Salmonella

Abstract A study was conducted to determine the efficacy of detecting low contamination levels of Salmonella in peanut butter using Assurance GDS for Salmonella (GDS). This study was conducted under the AOAC Research Institute Emergency Response Validation program. A set of samples was each analyzed by the GDS and the U.S. Food and Drug Administration's Bacteriological Analytical Manual reference method. Among the 45 total samples and controls, 26 samples were positive by the GDS and 24 were positive by the reference method. Statistical analyses of these unpaired samples indicated that, for all levels analyzed, the differences between the GDS and reference method results were not statistically different at the 5 level.

TRANSIA PLATE Salmonella Gold

A study was conducted to determine the efficacy of detecting low contamination levels of Salmonella in peanut butter using TRANSIA PLATE Salmonella Gold (TPSG). This study was conducted under the AOAC Emergency Response Validation program. A set of samples was each analyzed by the TPSG method and the U.S. Food and Drug Administration's Bacteriological Analytical Manual reference method. Among the 45 total samples and controls, 26 were positive by the TPSG and 24 were positive by the reference method. Statistical analyses of these unpaired samples indicated that for all levels analyzed, the difference between the TPSG and reference method results were not statistically different at the 5% level.

GeneQuence Salmonella Assay

A study was conducted to validate the GeneQuence Salmonella DNA hybridization assay, Performance Tested Method 030201, for detection of Salmonella spp. in peanut butter. The study was organized by the AOAC Research Institute under its Emergency Response Validation program. Peanut butter samples inoculated with S. Typhimurium were prepared by an independent laboratory and shipped to study participants for testing. The set of blind-coded test samples consisted of five uninoculated controls, 20 portions inoculated with S. Typhimurium at a low level [determined by most probable number (MPN) analysis to contain 1.1 CFU/25 g portion], and 20 portions inoculated with S. Typhimurium at a higher level (11 CFU/25 g portion by MPN analysis). Samples were tested in parallel by the GeneQuence method and by the U.S. Food and Drug Administration's Bacteriological Analytical Manual reference culture procedure. All five control samples were negative by both methods. For the low-level samples, the same two samples were positive by both the GeneQuence and reference methods. For the high-level samples, the same 19 samples were positive by both methods. All positive GeneQuence assays were confirmed by plating from associated broth cultures. Agreement between the GeneQuence and reference methods was 100%. Sensitivity and specificity of the GeneQuence method were both 100%. Because neither the low- nor the high-level samples yielded the desired fractional positive results (5-15 positives out of 20 samples), a second trial was conducted. Samples in the second trial contained 0.1 and 0.5 CFU/25 g portion for the low and high levels, respectively. All five control samples were negative by both methods. For the low-level samples, the same two samples were positive by both the GeneQuence and reference methods. For the high-level samples, the same three samples were positive by both methods. All positive GeneQuence assays were confirmed by plating from associated broth cultures. Sensitivity and specificity of the GeneQuence method were both 100%. Although once again the desired level of fractional positive results was not obtained, there was 100% agreement between the GeneQuence and reference methods. Based on the results of both trials, it is recommended that the validated claims for Performance Tested Method 030201 be expanded to include peanut butter.