Gram-negative identification card for identification of Salmonella, Escherichia coli, and other Enterobacteriaceae isolated from foods: collaborative study

Twelve laboratories evaluated the Gram-Negative Identification (GNI) Card to identify members of the Enterobacteriaceae. Eighty-four isolates, previously isolated from foods, were used in the collaborative study; the isolates represented 12 genera within the Enterobacteriaceae group. Each collaborator streaked each isolate on tryptic soy agar plates for purity. In the method, plates are incubated 18-24 h at 35 degrees C. Isolated colonies are then subcultured to tryptic soy agar slants and incubated 18-24 h at 35 degrees C. An emulsion is made from the growth on the slant in 1.8 mL 0.45% sodium chloride solution. The GNI Card is filled and placed in a reader/incubator. Isolates are identified and an identification is printed. The Vitek System correctly identified 96.7% of Salmonella sp., 97.0% of Escherichia coli, and an average of 93.8% of the other enteric genera. The method using the Vitek System and GNI Card has been approved interim official first action by AOAC as a screening method for the presumptive identification of Salmonella sp., E. coli, and other Enterobacteriaceae isolated from foods.     

Fluorogenic assay for rapid detection of Escherichia coli in chilled and frozen foods: collaborative study

A collaborative study was conducted to compare a proposed LST-MUG method with the AOAC official method for Escherichia coli detection. E. coli produces an enzyme, beta-glucuronidase, which cleaves the substrate, 4-methyl-umbelliferyl-beta-D-glucuronide (MUG), to yield a fluorescent end product. Incorporation of the MUG substrate into lauryl tryptose broth (LST) enables a rapid quantitative method for screening E. coli, which is detected by fluorescence of the medium under longwave UV light. In this collaborative study, 5 food samples, 2 frozen (entree sauce/gravy and dairy topping) and 3 chilled (hamburger, pork sausage, and cheese), were tested for E. coli detection by 17 collaborating laboratories. Results indicate that the LST-MUG method is equal to or better than the current AOAC method for detecting E. coli. The LST-MUG method has been adopted official first action.     

Dry rehydratable film for enumeration of total coliforms and Escherichia coli in foods: collaborative study

Rehydratable dry-film plating methods for total coliforms and Escherichia coli in foods have been compared to the AOAC most probable number methods. Fourteen laboratories participated in the collaborative study. Three coliform and E. coli levels in 6 samples of 4 product types (flour, nuts, cheese, and beef with gravy) and in 3 samples of 2 product types (mushrooms and raw turkey) were tested in duplicate by the participants. The mean log counts for the 3 methods were comparable. In general, the repeatability and reproducibility variances of the plating methods were as good as or better than that of the MPN method. The method has been adopted official first action by AOAC.     

Enzyme immunoassay for detection of Salmonella in foods: collaborative study

A collaborative study was performed in 25 laboratories to validate an enzyme immunoassay (EIA) procedure utilizing 2 specific monoclonal antibodies for rapid detection of Salmonella in foods. The EIA was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy isolate, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed, with the exception of poultry which was naturally contaminated. There was no significant difference in the productivity of the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been adopted official first action.     

Visual immunoassay for detection of Salmonella in foods: collaborative study

A collaborative study was performed in 13 laboratories to validate a visual enzyme immunoassay (EIA) procedure, TECRA, for rapid detection of Salmonella in foods. The EIA method was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. There was no significant difference in the productivity of the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been approved interim official first action.     

DNA hybridization assay for detection of Salmonella in foods: collaborative study

A collaborative study was performed in 11 laboratories to validate a DNA hybridization (DNAH) procedure for detection of Salmonella in foods. The DNAH procedure was compared to the standard culture method for detection of Salmonella in 6 foods: ground pepper, soy flour, dry whole egg, milk chocolate, nonfat dry milk, and raw deboned turkey. With the exception of turkey which was naturally contaminated, uninoculated and inoculated samples of each food group were analyzed. Results for the DNAH method were significantly better than for the standard culture method at the 5% probability level for the detection of Salmonella in turkey. There was no significant difference between the methods for the other 5 foods. The method has been adopted official first action.     

Enzyme immunoassay for detection of Salmonella in low-moisture foods: collaborative study

A collaborative study was performed in 15 laboratories to evaluate a modification of the enzyme immunoassay (EIA) method for detection of Salmonella in foods (46.B21-46.B29). The modified EIA requires 18-24 h pre-enrichment, 6-8 h selective enrichment, and 14-18 h M-broth post-enrichment prior to performing the assay, which requires 1-2 h. Total assay time is 40-52 h. The modified method was compared with the standard culture method for detection of Salmonella in 5 low-moisture foods: nonfat dry milk, milk chocolate, meat and bone meal, dry whole egg, and ground pepper. The modified method has been adopted official first action for use with low-moisture foods.     

Fluorescent enzyme immunoassay for rapid screening of Salmonella in foods: collaborative study

A collaborative study was performed in 13 laboratories to validate an enzyme immunoassay (EIA) procedure for rapid detection of Salmonella in foods. The EIA was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been adopted official first action as a rapid screening method for detection of Salmonella.     

Immunodiffusion screening method for detection of motile Salmonella in foods: collaborative study

A collaborative study was performed to validate the performance of the 1-2 TEST for detection of motile salmonellae in foods. Detection is based on observation of an immobilized band of cells. Twenty-three laboratories participated in the study. The 1-2 TEST (immunodiffusion test) was compared with the standard culture procedure (BAM/AOAC; FDA Bacteriological Analytical Manual) for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole egg, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. After the tests on the 6 foods were completed, analysis of the data for turkey and soy flour showed that certain collaborators obtained data inconsistent with the data from the majority of collaborators. No specific method deviations to account for the inconsistencies were reported by those collaborators, so the collaborative testing of these 2 foods was repeated. Analysis of data for pepper, chocolate, nonfat dry milk, dried whole egg, and the second set of soy flour and turkey indicated 96.1% agreement between the BAM/AOAC and immunodiffusion test methods. The false negative rates for the immunodiffusion test and BAM/AOAC methods were 3.6 and 1.7%, respectively. There was no significant difference in the productivity of the immunodiffusion test and BAM/AOAC method at the 5% level for any of the 6 foods. The immunodiffusion screening method has been approved official first action for detection of motile Salmonella in foods.     

Enumeration of total coliforms, fecal coliforms, and Escherichia coli in foods by hydrophobic grid membrane filter: collaborative study

A collaborative study was conducted in 18 laboratories to assess the performance of the hydrophobic grid membrane filter method against that of the AOAC official first action method 46.013-46.016 for enumerating total and fecal coliforms and Escherichia coli. The study was carried out on frozen breaded fish, raw comminuted poultry, unroasted walnut pieces, ground black pepper, and cheddar cheese. The hydrophobic grid membrane filter method recovered significantly larger numbers of target bacteria in 7 of the food/analysis combinations: fecal coliforms in fish; E. coli in poultry; fecal coliforms and E. coli in walnuts; and total coliforms, fecal coliforms and E. coli in black pepper. Random error (Sr2) associated with the hydrophobic grid membrane filter method was significantly lower than that of the reference method in over 30% of the paired sample series. The hydrophobic grid membrane filter method for total coliform, fecal coliform, and E. coli enumeration in foods has been adopted official first action.     

Hydrophobic grid membrane filter/MUG method for total coliform and Escherichia coli enumeration in foods: collaborative study

Twenty-four laboratories participated in a collaborative study to validate a hydrophobic grid membrane filter (HGMF) method incorporating the use of 4-methylumbelliferyl-beta-D-glucuronide (MUG) for enumeration of total coliform and Escherichia coli bacteria in foods by comparing its performance against the AOAC 3-tube MPN method (46.013-46.016). Raw milk, raw ground poultry, whole egg powder, cheese powder, and ground black pepper were included in the study. The total coliform methods did not differ significantly, except that the 3-tube method detected a significantly higher level of total coliforms than did the HGMF method in the ground black pepper. Conversely, the HGMF/MUG E. coli method detected significantly higher numbers of E. coli present in the egg powder, cheese powder, and ground black pepper samples, while not differing significantly from the 3-tube method for the raw milk and raw ground poultry samples. The overall confirmation rate of MUG-positive colonies isolated using the HGMF method was 99.5%. The hydrophobic grid membrane filter/MUG method has been adopted official first action as an additional method to AOAC official final action method 46.030-46.034.     

Colorimetric deoxyribonucleic acid hybridization assay for rapid screening of Salmonella in foods: collaborative study

A collaborative study was performed in 11 laboratories to validate a colorimetric DNA hybridization (DNAH) method for rapid detection of Salmonella in foods. The method was compared to the standard culture method for detection of Salmonella in nonfat dry milk, milk chocolate, soy isolate, dried whole egg, ground black pepper, and raw ground turkey. Samples inoculated with high (0.4-2 cells/g) and low (0.04-0.2 cells/g) levels of Salmonella and uninoculated control samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by DNAH and culture procedure for any of the 6 foods. The colorimetric DNA hybridization assay screening method has been adopted official first action as a rapid screening method for detection of Salmonella in all foods.     

Improved hydrophobic grid membrane filter method, using EF-18 agar, for detection of Salmonella in foods: collaborative study

A collaborative study was carried out in 30 laboratories to validate improvements to the official final action hydrophobic grid membrane filter (HGMF) screening method for Salmonella in foods, 985.42, by comparing the performance of the improved HGMF method against that of the AOAC/BAM conventional culture method. Six products were included in the collaborative study: milk chocolate, raw deboned poultry meat, black pepper, soy flour, egg yolk powder, and nonfat dry milk. The raw deboned poultry meat was naturally contaminated with Salmonella, and the remaining 5 products were each inoculated in advance with low levels of individual Salmonella serotypes. The AOAC/BAM method produced 11 false negative results and the improved HGMF method produced 18 false negative results. The improved HGMF Salmonella method has been approved interim official first action for all foods to replace the HGMF official final action method, 985.42.     

Comparative accuracy of five biochemical systems for identifying Salmonella and related foodborne bacteria: collaborative study.

The comparative accuracy of 4 biochemical diagnostic kits (API, Enterotube, Minitek, and Pathotec) and the conventional (AOAC) tube system for identifying primarily Salmonella and other enteric isolates was collaboratively studied. Each of 11 participating analysts received 40 foodborne isolates (25 Salmonella and 15 non-Salmonella cultures), representing a total of 440 cultures examined by each identification system. In decreasing order of accuracy, the overall number of correctly identified cultures with each of the systems was as follows: AOAC, 423 (96.1%), Minitek, 403 (91.6%), Enterotube, 395 (89.8%), API, 394 (89.5%), and Pathotec, 373 (84.8%). A cost analysis showed that all 4 diagnostic kit systems were less expensive than the conventional AOAC tube system for a single culture identification. Three of the diagnostic kits have been adopted as official first action as alternatives to the AOAC biochemical tube system for presumptive generic identification of foodborne Salmonella and for screening and eliminating non-Salmonella isolates. Routine incorporation of any one of the 3 diagnostic kits, however, should be preceded by the demonstration in the analyst's own laboratory of adequate correlation between the kit and the AOAC system.     

Defined substrate technology method for rapid and specific simultaneous enumeration of total coliforms and Escherichia coli from water: collaborative study

The defined substrate technology (DST) method is a reagent system designed to enumerate specific target microbes(s) from a mixture of bacteria. The system simultaneously enumerates total coliforms and Escherichia coli directly from a water sample. The reagent contains o-nitrophenyl-beta-D-galactopyranoside (ONPG), which is hydrolyzed by total coliforms to produce a yellow chromogen, and 4-methylumbeilliferyl-beta-D-glucuronide (MUG), which is hydrolyzed and fluoresces when E. coli organisms grow. Noncoliform bacteria are suppressed and cannot metabolize the indicator nutrients. Nine laboratories participated in a field evaluation of the method, which covered a wide range of surface and subsurface water sources and water-processing modalities, including the examination of natural samples. The DST system was compared to multiple-tube fermentation (MTF) (quantitative) and presence-absence (P-A) (qualitative) Standard Methods formats. Comparison of water samples from natural sources by using the most probable number (MPN) procedure showed that the DST test was equivalent to the currently used MTF test. Results from the DST and the qualitative P-A procedure showed that these tests agreed with each other in 94% of the water samples analyzed. Specificity of the DST method was established by subculturing a species consistent with a total coliform or E. coli from each positive tube. Eight laboratories participated in a collaborative study of the method. Each laboratory received 3 concentrations of E. coli (organisms/100 mL); 10 (low); 60 (medium); and 120 (high). The DST test was inoculated from a split sample of each bacterial density in parallel with Standard Methods brilliant green lactose broth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme immunoassay for determination of gluten in foods: collaborative study

A collaborative study was performed in 15 laboratories to validate a monoclonal antibody-based enzyme immunoassay (EIA) for determination of gluten in foods. The study included 13 samples: maize starch, "gluten-free" baking mixes, wheat flours, cookies, cooked meats, and a soup. Gluten was present in these samples at either zero or 0.02 to 10% by weight, i.e., over almost 3 orders of magnitude. The mean assay values for the foods varied from 88 to 105% of the actual amounts. The assay was quantitative for cereal products and the soup with repeatability (RDS-r, relative standard deviation) and reproducibility (RSD-R) of 16-22% and 24-33%, respectively. The assay was semiquantitative for the processed meat products (RSD-r 14 and 26% and RSD-R 46 and 56%), probably because gluten was unevenly distributed in the small (1 g) samples that were analyzed. The ELISA method produced no false positive results, and false negatives obtained with tannin-containing foods could be avoided by use of a modified sample extractant. None of the collaborators reported problems in following the protocol. The method has been adopted official first action by AOAC for determination of wheat gluten in foods.     

Anion-exchange method for determination of phytate in foods: collaborative study

Phytate, a naturally occurring organic compound found in plant seeds, roots, and tubers, was determined in a collaborative study using a modified anion-exchange method. Seven samples (peanut flour, oats, rice, isolated soybean protein, a vegetarian diet composite, wheat bran, and whole wheat bread), supplied as blind duplicate samples, were analyzed in triplicate by 7 collaborators. Phytate concentrations in the samples ranged from 2.38 to 46.70 mg/g. Relative standard deviations (RSD = CV) for repeatability ranged from 2.5 to 10.1%, and for reproducibility, from 4.5 to 11.0%. The method has been adopted official first action.     

Optimized Monier-Williams method for determination of sulfites in foods: collaborative study

A collaborative study was conducted of the Food and Drug Administration (FDA)-optimized Monier-Williams method for determining sulfites in foods. Twenty-one industry and government laboratories participated in the study, which was jointly sponsored by the National Food Processors Association and FDA. Familiarization samples were shipped to each collaborator. Collaborators were permitted to proceed to the main study only after they demonstrated ability to perform the method to ensure that the study tested the performance of the method itself and not that of the individual laboratories. The study design involved 3 food matrixes (hominy, fruit juice, and protein [seafood]). Each matrix was prepared at 3 sulfite levels--the regulatory level, half the regulatory level, twice the regulatory level--and as a blank. All test samples were analyzed as blind duplicates, which gave each collaborator a total of 24 test portions. Collaborative recoveries gave a reproducibility (among-laboratories) coefficient of variation that ranged from 15.5 to 26.6% for sulfite determined as SO2 by weight in the 3 foods at the 10 ppm level. The optimized Monier-Williams method has been approved interim official first action to replace the AOAC modified Monier-Williams method, 20.123-20.125.     

Enumeration and confirmation of Bacillus cereus in foods: collaborative study

A collaborative study was conducted in 15 laboratories to evaluate 2 different techniques for enumerating Bacillus cereus in foods. A direct plating technique using mannitol-egg yolk-poly-myxin agar and a most probable number (MPN) technique using trypticase-soy-polymyxin broth were compared for the enumeration of high and low populations of B. cereus in mashed potatoes. The collaborative results showed that the overall mean recovery obtained with the low population level was essentially the same by both techniques. However, the overall mean recovery was significantly higher by the direct plating technique at the high population level. A statistical evaluation of the data also showed that the direct plating technique had better repeatability and reproducibility than did the MPN technique at both the high and low population levels. These results suggest that the MPN technique is suitable for examining foods containing low populations of B. cereus, but that the direct plating technique is preferable for foods that contain a high population of this organism. The confirmatory technique used in the proposed method is reliable for presumptive identification of isolates as B. cereus. The method has been adopted as official first action.