Effect of sample preparation and preenrichment media on the recovery of Salmonella from cantaloupes, mangoes, and tomatoes

Studies were conducted to determine the relative effectiveness of buffered peptone water (BPW), lactose (LAC) broth, and Universal Preenrichment (UP) broth for the recovery of Salmonella organisms from fruit rinses, whole fruit, and comminuted fruit. In the first phase, the relative effectiveness of the rinse and soak methods for the recovery of Salmonella from surface-contaminated mangoes and tomatoes was examined. Fruits were spot inoculated with single Salmonella serovars and held for 4 days at 2-6 degrees C before analysis was initiated. The contaminated fruit was rinsed in portions of BPW, LAC broth, or UP broth. Portions from each rinse were added to its respective broth (e.g., BPW to BPW). Individual whole fruit, in their remaining broth rinses (soak method), and the fruit rinse/broths (rinse method) were incubated for 24 h at 35 degrees C. The Bacteriological Analytical Manual (BAM) Salmonella culture method was followed thereafter. The soak method produced significantly greater numbers (P < 0.05) of positive test portions than did the rinse method for the analysis of mangoes (93 versus 12) and tomatoes (85 versus 34). The 3 broths were comparable for the recovery of Salmonella for both the soak and the rinse methods for mangoes. For tomatoes, there were no significant differences among the broths for the soak method, but BPW and UP broth were significantly more productive (P < 0.05) than LAC broth by the rinse method. In the second phase, the relative effectiveness of LAC broth, BPW, and UP broth for the recovery of Salmonella from comminuted fruit was examined. Fruits were contaminated with single Salmonella serovars and aged for 4 days at 2-6 degrees C. Twenty 25 g test portions were preenriched in each of the following broths: BPW, LAC broth, and UP broth. The BAM Salmonella culture method was followed thereafter. For cantaloupes, significantly more (P < 0.05) Salmonella-positive test portions were recovered with UP broth (96 Salmonella-positive test portions) and BPW (87 Salmonella-positive test portions) than with LAC broth (57 Salmonella-positive test portions). For mangoes, BPW recovered an arithmetically larger number of Salmonella-positive test portions (27 Salmonella-positive test portions) than did either LAC broth (14 Salmonella-positive test portions) or UP broth (18 Salmonella-positive test portions). For tomatoes, there were no significant differences among the broths: BPW recovered 65 Salmonella-positive test portions, UP broth recovered 62 Salmonella-positive test portions, and LAC broth recovered 60 Salmonella-positive test portions. For the analysis of whole fruit, it is recommended that the soak method be used. For whole fruit analyzed with the soak method, UP broth should be used for tomatoes and BPW should be used for mangoes. It is further recommended that UP broth be used for the analysis of comminuted cantaloupes and that BPW be used for the analysis of comminuted mangoes and tomatoes.     

Effectiveness of universal pre-enrichment broth for recovery of Salmonella from selected dairy foods

The relative efficiencies of 2 Bacteriological Analytical Manual (BAM) pre-enrichments, lactose broth (LAC) and brilliant green water (BGW), were compared with Universal Pre-enrichment (UP) broth for the recovery of individual Salmonella serovars from instant nonfat dry milk (NFDM), dry whole milk (DWM), lactic casein (LC), and liquid whole milk (LWM). BGW was compared with UP broth for the analysis of NFDM and DWM but not with the other 2 matrixes. LAC was compared with UP broth for the analysis of LC and LWM. UP broth was made both from a commercial dehydrated preparation (UPC) and from individual ingredients (UPI). Bulk quantities of the selected dairy foods were inoculated with Salmonella serovars at levels intended to produce fractionally positive results, where at least half of the test portions analyzed, with one of the methods being evaluated, would be shown to be Salmonella-positive. For NFDM, in 6 of 9 experiments, with 2 different Salmonella serovars, BGW was significantly more productive than either UPI or UPC broth (p < 0.05). Salmonella was recovered from 118 of 180 test portions with BGW, from 25 of 180 test portions with UPC, and from 14 of 180 test portions with UPI. For DWM, in 2 of 4 experiments, with 2 different Salmonella serovars, BGW was significantly more productive than either UPI or UPC broth (p < 0.05). Salmonella was recovered from 67 of 80 test portions with BGW, from 36 of 80 test portions with UPC, and from 37 of 80 test portions with UPI. For LWM, in 9 of 9 experiments, with 3 different Salmonella serovars, there were no significant differences among the broths. Salmonella was recovered from 120 of 180 test portions with LAC, from 135 of 180 test portions with UPC, and from 129 of 180 test portions with UPI. For LC, in 5 of 7 experiments, with 2 different Salmonella serovars, both UPI and UPC broth were significantly more productive than LAC (p < 0.05). Salmonella was recovered from 42 of 140 test portions with LAC, from 114 of 140 test portions with UPC, and from 114 of 140 test portions with UPI. In addition, overall results showed that UPC and UPI broths were equivalent for the recovery of Salmonella from the foods tested, without regard to their performance in comparison with either LAC or BGW.     

Enhanced recovery of Salmonella from apple cider and apple juice with universal preenrichment broth

A comparison was made of the relative efficiencies of Universal Preenrichment (UP) broth and lactose broth for the recovery of a variety of Salmonella serovars from pasteurized and unpasteurized apple cider and pasteurized apple juice. Bulk portions of juice were contaminated with single Salmonella serovars at high and low levels of 0.4 and 0.04 CFU/mL, respectively. The juice was aged for a minimum of 5 days at 2-5 degrees C. On the day analysis was initiated, each of 20 test portions (25 mL) of the contaminated juice was preenriched in UP broth and in lactose broth. The Bacteriological Analytical Manual Salmonella culture method was followed thereafter. For pasteurized apple cider, UP broth recovered significantly (p < 0.05) more Salmonella-positive test portions than did lactose broth (112 and 75, respectively). For unpasteurized apple cider, UP broth recovered significantly more Salmonella-positive test portions than did lactose broth (326 and 221, respectively). For pasteurized apple juice, UP broth recovered more Salmonella-positive test portions than did lactose broth (93 and 81, respectively). However, this difference was not statistically significant. These results indicate that UP broth should replace lactose broth for the analysis of pasteurized and unpasteurized apple cider and pasteurized apple juice.     

The effect of preenrichment and selective enrichment media on recovery of Salmonella Typhi from the tropical fruit mamey

The Bacteriological Analytical Manual (BAM) Salmonella culture method did not detect Salmonella Typhi in mamey, the tropical fruit that was implicated in a 1999 typhoid outbreak. The relative effectiveness of BAM's nonselective preenrichment and selective media for the recovery of S. Typhi from mamey was examined to determine if the BAM's preenrichment/selective enrichment strategy was the cause of the method's failure with this food. The preenrichment media were lactose broth, buffered peptone water (BPW), and universal preenrichment (UP) broth. The selective enrichment media were selenite cystine (SC) broth, tetrathionate (TT) broth, and Rappaport-Vassiliadis (RV) medium. UP broth was significantly more effective (P < 0.05) than either lactose broth or BPW for the recovery of 2 different S. Typhi strains from mamey. Of 120 test portions tested, 105 S. Typhi-positive test portions were recovered using UP broth, whereas only 1 S. Typhi-positive test portion was recovered using BPW, and no S. Typhi-positive test portions were recovered using lactose broth. SC and TT broths were both significantly more effective (P < 0.05) than RV medium. Of 105 S. Typhi-positive test portions, SC broth recovered 80, TT broth recovered 67, and RV medium recovered 9. After the above comparison, an incomplete UP (UPI) broth formulation was found to be significantly more effective (P < 0.05) than the complete formulation (UPC). Of 80 total positive test portions, UPI recovered 71, whereas UPC recovered only 48. The following UP broth formulations were compared to determine if any of the components of the UP broth formulation were inhibitory to S. Typhi: UPC, UP broth without sodium pyruvate (UPS), UP broth without ferric ammonium citrate (UPF), UP broth without MgSO4 (UPM), and UPI. It was found that none of the ingredients were inhibitory to S. Typhi and that, out of 140 total test portions, UPI and UPF, with 108 and 103 positive test portions, respectively, were significantly more effective (P < 0.05) than the other UP broth formulations (82 for UPC, 74 for UPS, and 60 for UPM). Rather than being inhibitory to the growth of S. Typhi, it appears that ferric ammonium citrate enhanced the growth of competitors which suppressed the growth of S. Typhi. These results demonstrate that UPI and UPF are effective for the recovery of S. Typhi from mamey.     

In-house validation study for salmonella unique testing of juice (modification of AOAC official method 2000.07)

Following an industry request, a study was undertaken to validate a minor change to the Unique method for testing fruit juice. Twenty foods were tested in the original precollaborative study for TECRA Unique Salmonella test (2000.07). To validate the modification for juice, both the modified method (42 degrees C module incubation with a 5 h replication step) and the current AOAC Method 2000.07 (37 degrees C incubation with a 4 h replication step) were compared with the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM; 8th Ed., 1998) reference method, which uses lactose broth as pre-enrichment medium. Twenty uninoculated replicates, 20 replicates with low-level inoculum (target 1-5 cells/25 g), and 20 replicates with high-level inoculum (target 10-50 cells/25 g) were tested for a single batch of fresh orange juice in accordance with AOAC requirements. There was exact agreement between the 2 Unique methods for all samples and exact agreement between the 2 Unique methods and the BAM method for the uninoculated and high-level inoculum samples. For low-level inoculum, 17 samples were confirmed positive with the new Unique method, 17 with AOAC Method 2000.07, and 14 with the BAM method.     

Comparison of assurance gold salmonella EIA, BAX for screening/Salmonella, and GENE-TRAK Salmonella DLP rapid assays for detection of Salmonella in alfalfa sprouts and sprout irrigation water

The Assurance Gold Salmonella EIA, BAX for Screening/Salmonella, and GENE-TRAK Salmonella DLP rapid assays were compared with official cultural methods described in the Bacteriological Analytical Manual (BAM) for analysis of alfalfa sprouts and sprout irrigation water for the presence of Salmonella. The lower limits of detection of 4 serovars of Salmonella cells (S. tennessee, S. muenchen, S. mbandanka, and S. cubana) in pure culture were determined as approximately log10 2, 5, and 6 for the BAX, GENE-TRAK, and Gold EIA, respectively. Despite its low detection limit, the BAX did not perform as well as the other assays in analyzing contaminated sprouts and sprout irrigation water. For 4 different lots of sprouts and sprout irrigation water samples inoculated with the 4 serovars at low [1-2 colony forming units (CFU/g)] and high (68-180 CFU/g) levels, the BAX detected Salmonella in 58/64 (90.6%) of the samples, compared with 64/64 (100%) by the GENE-TRAK, Gold EIA, and BAM methods. Assay performance was also compared for analysis of naturally contaminated sprouts and sprout irrigation water with 3 lots of alfalfa sprouted seeds associated with different salmonellosis outbreaks. Positive assay results for the naturally contaminated samples were Gold EIA 41, GENE-TRAK 36, BAM 33, and BAX 13.     

Evaluation of culture media for selective enrichment and isolation of Salmonella in seafood

Seafood, including fish, shrimp, clam, crab, mussel, oyster, lobster, squid, octopus, and cuttlefish samples, was used to compare the recovery of Salmonella serovars by different selective enrichment and isolation media. The samples were selectively enriched in Rappaport-Vassiliadis (RV) broth and tetrathionate broth (TT), followed by selective isolation on Hektoen enteric (HE) agar, xylose lysine desoxycholate (XLD) agar, bismuth sulfite (BS) agar, and Brilliant Green (BG) agar media. Of 443 seafood samples analyzed, 108 were found to be contaminated with Salmonella. The role of selective enrichment in Salmonella spp. recovery with RV medium was distinctly high (70%) compared to TT broth (30%). The selective enrichment in RV broth followed by selective isolation on XLD, HE, BS, and BG agar recovered Salmonella at levels of 56, 41, 28, and 16%, respectively. Similarly, after enrichment in TT broth, XLD and HE agars recovered 27 and 23% respectively. The recovery of Salmonella with enrichment in TT followed by isolation on BS and BG was abysmally low at 4.6 and 5%, respectively. There was no significant difference (P > 0.05) in the recovery of Salmonella using the combinations of XLD and HE media with selective enrichment in RV broth. However, performance difference (P < 0.05) was observed in the recovery when BS and BG with RV, and XLD, HE, BS, and BG agars with TT broth were used. The present study showed that the combination of RV with XLD was the most efficient media for isolation of Salmonella from seafood when compared to other isolation media combinations.     

Evaluation of sample preparation methods for the isolation of Salmonella from alfalfa and mung bean seeds with the Bacteriological Analytical Manual's Salmonella culture method

Five pre-enrichment methods were evaluated for effectiveness with the U.S. Food and Drug Administration's Bacteriological Analytical Manual Salmonella culture method in recovering S. Stanley, S. Poona, and S. Muenchen from artificially contaminated alfalfa seeds, and S. Saintpaul, S. Anatum, and S. Infantis from artificially contaminated mung bean seeds. The methods included: (1) Soak.--Test portions were inoculated into pre-enrichment media; (2) Rinse.--Test portions were rinsed with pre-enrichment media, and the media was decanted from the test portions; (3) Rinsed seed.--Pre-enrichment media was added to the test portions that were rinsed in the rinse method; (4) Wet blend.--Test portions were blended with the pre-enrichment media; and (5) Dry blend.--Test portions were blended prior to pre-enrichment. The methods of pre-enrichment were also evaluated for effectiveness in recovering Pantoea agglomerans from alfalfa and mung bean seeds with a modified culture method for the recovery of Enterobacteriaceae from foods. The purpose of these studies was to provide a model for the recovery of Salmonella that may occur in seeds as a natural contaminant. The relative effectiveness of the soak method was consistently superior to the rinse method in isolating the selected Salmonella serovars from both seed types. Statistically, the rinsed seed method was as effective as the soak method in all trials, except 1 of 3, with S. Muenchen and alfalfa seeds (P > 0.05). The relative effectiveness of the methods in isolating P. agglomerans from alfalfa and mung bean seeds was similar to that observed with the artificially contaminated test portions. The soak method was consistently the most effective method and the rinse method was consistently the most ineffective method. The rinsed seed, wet blend, and dry blend methods were also as effective as the soak method in all 3 trials with each seed type (P > 0.05).     

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

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

Validation study to demonstrate the equivalence of a minor modification (TECRA ULTIMA protocol) to AOAC Method 998.09 (TECRA Salmonella Visual Immunoassay) with the cultural reference method

The TECRA Salmonella Visual Immunoassay (VIA) using Rappaport-Vassiliadis RV[R10] as a single selective enrichment broth has Final Action approval (AOAC Method 998.09). TECRA has recently developed a protocol (TECRA ULTIMA), which involves the addition of a new additive to a 1 mL aliquot of the RV[R10] broth, prior to the heat-killing step, thereby allowing the RV[R10] broth to be tested directly in the kit and thus eliminating the need for the 2 h post-enrichment in M broth. An in-house validation study was conducted to compare the modified AOAC Method 998.09 to the reference culture method. Three foods were used in the study: Naturally contaminated raw ground poultry at high (10-50 cells/25 g), and low (1-5 cells/25 g) levels; and milk powder and peanut butter, artificially inoculated at low and high levels with Salmonella bovismorbificans and S. enterica Mbandaka, respectively. Twenty test portions were analyzed for each level with 10 uninoculated control samples per food. Overall, no significant differences (p <0.05) were observed when the proportion of positive test portions for the modified VIA were compared with that for the reference method. This minor modification, which employs the additive (provided in the TECRA ULTIMA SALMONELLA Test Kit) to permit the direct analysis of RV[R10] broth has demonstrated the utility of the TECRA ULTIMA SALMONELLA protocol. It is recommended that the minor modification to Method 998.09 be approved First Action as an additional option within the method.     

Evaluation of VIDAS Salmonella (SLM) immunoassay method with Rappaport-Vassiliadis (RV) medium for detection of Salmonella in foods: collaborative study

A collaborative study was conducted to compare the VIDAS Salmonella (SLM) with Rappaport-Vassiliadis (RV) method for detection of Salmonella in foods to the current standard method presented in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) and the culture method presented in AOAC's Official Methods of Analysis. The VIDAS SLM with RV method uses tetrathionate broth in combination with RV medium in place of selenite cystine broth for selective enrichment, thereby eliminating the hazardous waste issue for laboratories. Twenty five laboratories participated in the evaluation, each testing one or more of 8 test products: nonfat dry milk, dried egg, soy flour, lactic casein, milk chocolate, raw ground pork, raw ground turkey, and raw peeled shrimp. Results of the study showed no significant differences in the numbers of confirmed positive samples with the VIDAS SLM with RV procedure and the BAM/AOAC culture procedure. The VIDAS SLM with RV method was effective for rapid detection of Salmonella in foods. It is recommended that AOAC INTERNATIONAL modify the VIDAS Salmonella SLM procedure to include the RV method.     

Multiplex sorting of foodborne pathogens by on-chip free-flow magnetophoresis

This study reports multiplex sorting of Salmonella typhimurium and Escherichia coli 0157, from broth cultures and from pathogen-spiked skinned chicken breast enrichment broths by employing microfluidic free-flow magnetophoresis. Magnetic beads of different sizes and magnetite content, namely Dynabeads anti-salmonella and Hyglos-Streptavidin beads together with the corresponding pathogen-specific biotinylated recombinant phages, were utilised as affinity solid phases for the capture and concentration of viable S. typhimurium and E. coli 0157. Following optimisation, the protocol was used to demonstrate continuous magnetophoretic sorting of the two pathogen-bound magnetic bead populations from mixed cultures and from pathogen-spiked chicken pre-enrichment broths under the influence of a Halbach magnet array. For example, in the latter case, a pure population of S. typhimurium-bound Dynabeads (72% recovery) was sorted from a 100 μL mixture containing E. coli 0157-bound Hyglos beads (67% recovery) within 1.2 min in the presence of 0.1% Tween 20. This proof-of-principle study demonstrates how more than one pathogen type can be simultaneously isolated/enriched from a single food pre-enrichment broth (e.g. Universal food enrichment broth).     

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

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

Evaluation of VIDas immuno-concentration Salmonella/VIDAS salmonella immunoassay method for detection of Salmonella in selected foods: collaborative study

The VIDAS Immuno-concentration Salmonella ICS)/VIDAS Salmonella (SLM) immunoassay method for the detection of Salmonella was compared to the Bacteriological Analytical Manual (BAM)/AOAC culture method in a collaborative study. Thirty-two laboratories participated in the evaluation. Each laboratory tested one or more of the 6 test products: milk chocolate, nonfat dry milk, dried whole egg, soy flour, ground black pepper, and ground raw turkey. The 2 methods were in agreement for 1,266 of the 1,440 samples. Of the 174 samples not in agreement, 69 were VIDAS CS/SLM-positive and BAM/AOAC-negative and 105 were VIDAS ICS/SLM-negative and BAM/AOAC-positive.     

Evaluation of VIDAs Immuno-concentration Salmonella assay Plus selective plate method (Hektoen enteric,bismuth sulfite,Salmonella identification) for detection of Salmonella in selected foods: collaborative study

The VIDAS Immuno-concentration Salmonella (ICS) plus selective plate method (Hektoen enteric, bismuth sulfite, Salmonella identification) method for the detection of Salmonella was compared to the Bacteriological Analytical Manual (BAM)/AOAC culture method in a collaborative study. Thirty-two laboratories participated in the evaluation. Each laboratory tested one or more of the 6 test products: milk chocolate, nonfat dry milk, dried whole egg, soy flour, ground black pepper, and ground raw turkey. The 2 methods were in agreement for 1,283 of the 1,440 test samples. Of the 157 test samples not in agreement, 82 were VIDAS ICS plus selective plate-positive and BAM/AOAC-negative, and 75 were VIDAS ICS plus selective plate-negative and BAM/AOAC-positive.     

Rappaport-Vassiliadis medium for recovery of Salmonella spp. from low microbial load foods: collaborative study

Twenty-three laboratories participated in a collaborative study to compare the relative effectiveness of Rappaport-Vassiliadis (RV) medium incubated at 42 degrees C, selenite cystine (SC) broth (35 degrees C), and tetrathionate (TT) broth (35 and 43 degrees C) for recovery of Salmonella from the following foods with a low microbial load: dried egg yolk, dry active yeast, ground black pepper, guar gum, and instant nonfat dry milk. For dry active yeast, lauryl tryptose (LT) broth, incubated at 35 degrees C, was used instead of SC broth. All of the foods were artificially inoculated with single Salmonella serovars, that had been lyophilized before inoculation, at high and low target levels of 0.4 and 0.04 colony forming units/g food, respectively. For analysis of 870 test portions, representing all of the foods except yeast, 249 Salmonella-positive test portions were detected by RV medium, 265 by TT broth (43 degrees C), 268 by TT broth (35 degrees C), and 269 by SC broth (35 degrees C). For analysis of 225 test portions of yeast, 79 Salmonella-positive test portions were detected by RV medium, 79 by TT broth (43 degrees C), 84 by TT broth (35 degrees C), and 68 by LT broth (35 degrees C). RV medium was comparable to, or even more effective than, the other selective enrichments for recovery of Salmonella from all of the foods except guar gum. It is recommended that RV (42 degrees C) and TT (35 degrees C) be used with foods that have a low microbial load, except for guar gum for which SC (35 degrees C) and TT (35 degrees C) are recommended.     

Rapid methods for detection and enumeration of Campylobacter spp. in foods

Campylobacter spp. are the most commonly reported bacterial cause of acute diarrheal disease in humans throughout the world. Traditional cultural methods for the detection and quantitation of Campylobacterspp. are slow and tedious; therefore, specific, sensitive, and rapid methods for campylobacters are needed to collect sufficient data for risk assessment and food safety policy development. We developed several rapid methods based on polymerase chain reaction (PCR), DNA hybridization, hydrophobic grid membrane filters (HGMFs), and enzyme immunoassays (EIAs). A PCR assay targeting C. jejuni, combined with a simple sample preparation procedure, detects as few as 0.3 most probable number (MPN)/mL C. jejuni in naturally contaminated chicken rinses after 20-24 h enrichment. An HGMF-EIA method using a commercial polyclonal antibody for Campylobacter detects and enumerates thermophilic Campylobacter spp. from spiked chicken rinse and milk, and naturally contaminated chicken rinses. A C. jejuni-specific probe in an HGMF-DNA hybridization protocol specifically detects and quantitates C. jejuni in food samples. A dot-blot EIA combined with an MPN procedure quantitates thermophilic campylobacters from samples that might be difficult to filter through HGMFs.     

Salmonella in selected foods by VIDAS immuno-concentration Salmonella plus selective plate method (Hektoen enteric,xylose lysine desoxycholate,bismuth sulfite): collaborative study

The VIDAS Immuno-concentration Salmonella (ICS) plus selective plate method (Hektoen enteric, xylose lysine desoxycholate, bismuth sulfite) method for the detection of Salmonella was compared to the Bacteriological Analytical Manual (BAM)/AOAC culture method in a collaborative study. Thirty-two laboratories participated in the evaluation. Each laboratory tested one or more of the 6 test products: milk chocolate, nonfat dry milk, dried whole egg, soy flour, ground black pepper, and ground raw turkey. The 2 methods were in agreement for 1,297 of the 1,455 samples. Of the 158 samples not in agreement, 82 were VIDAS ICS plus selective plate-positive and BAM/AOAC-negative, and 76 were VIDAS ICS plus selective plate-negative and BAM/AOAC-positive.     

Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201

The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.     

Alternative anaerobic enrichments to the bacteriological analytical manual culture method for isolation of Shigella sonnei from selected types of fresh produce

Alternative methods of reducing oxygen during anaerobic enrichment in the Bacteriological Analytical Manual (BAM) Shigella culture method were evaluated and compared to the current and less practical GasPak method. The alternative anaerobic methods included the use of reducing agents in Shigella broth and reducing culture container headspace volume to minimize atmospheric effects on oxygen concentration in Shigella broth during enrichment. The reducing agents evaluated were sodium thioglycollate, L-cystine, L-cysteine, titanium(III) citrate, and dithiothreitol, each at concentrations of 0.1, 0.05, and 0.01%. The use of Oxyrase for Broth with the enrichment medium (Shigella broth) was evaluated at concentrations of 10, 20 and 30 microL/mL. Recoveries of chill- and freeze-stressed S. sonnei strains 357 and 20143 were determined with each anaerobic method, including the GasPak method, using inoculation levels ranging from 10(0)to 10(3) cells. For each anaerobic method, strain, inoculation level, and stress type, 5 replicate enrichments were evaluated by streaking to MacConkey agar for isolation. The numbers of cultures with each method from which S. sonnei was isolated were used to compare the alternative anaerobic methods to the GasPak method. The alternative anaerobic method with which chill- and freeze-stressed S. sonnei strains 357 and 20143 were isolated most consistently was the use of Oxyrase for Broth in Shigella broth at a concentration of 20 microL/mL. This method was compared to the GasPak anaerobic method in evaluations on the recovery of S. sonnei strains 357 and 20143 from artificially contaminated test portions of parsley, cilantro, green onions, strawberries, carrots, and celery. A third anaerobic method included the use of 0.5 cm mineral oil overlay on cultures containing Oxyrase for Broth at concentrations of 20 microL/mL. Recovery rates of strain 357 were significantly greater (p < 0.05) with the GasPak method than with Oxyrase for Broth, with and without the 0.5 cm mineral oil overlay, for test portions of parsley, cilantro, and celery. When Oxyrase for Broth was used with Shigella broth, strain 357 was isolated at higher rates from all produce types, except cilantro, when 0.5 cm mineral oil overlay was applied to enrichment cultures. The use of mineral oil overlay with Oxyrase for Broth also improved recovery of strain 20143 from test portions of all produce types except green onion and strawberries. These differences were significant (p < 0.05) with parsley, carrots, and cilantro (1 of 2 evaluations). No statistically significant differences (p > 0.05) between the GasPak and Oxyrase for Broth anaerobic methods occurred when mineral oil overlay was used with Oxyrase for Broth. The use of Oxyrase for Broth with a 0.5 cm mineral oil overlay is a practical alternative for anaerobic enrichment with the BAM method in the analysis of some produce types. Differences in recovery among the different produce types and methods occurred between S. sonnei strains 357 and 20143, emphasizing the need for additional S. sonnei strains in future evaluations.