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).

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.

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.

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.

Relative effectiveness of the Bacteriological Analytical Manual method for the recovery of Salmonella from whole cantaloupes and cantaloupe rinses with selected preenrichment media and rapid methods.

Soak and rinse methods were compared for the recovery of Salmonella from whole cantaloupes. Cantaloupes were surface inoculated with Salmonella cell suspensions and stored for 4 days at 2 to 6 degrees C. Cantaloupes were placed in sterile plastic bags with a nonselective preenrichment broth at a 1:1.5 cantaloupe weight-to-broth volume ratio. The cantaloupe broths were shaken for 5 min at 100 rpm after which 25-ml aliquots (rinse) were removed from the bags. The 25-ml rinses were preenriched in 225-ml portions of the same uninoculated broth type at 35 degrees C for 24 h (rinse method). The remaining cantaloupe broths were incubated at 35 degrees C for 24 h (soak method). The preenrichment broths used were buffered peptone water (BPW), modified BPW, lactose (LAC) broth, and Universal Preenrichment (UP) broth. The Bacteriological Analytical Manual Salmonella culture method was compared with the following rapid methods: the TECRA Unique Salmonella method, the VIDAS ICS/SLM method, and the VIDAS SLM method. The soak method detected significantly more Salmonella-positive cantaloupes (P < 0.05) than did the rinse method: 367 Salmonella-positive cantaloupes of 540 test cantaloupes by the soak method and 24 Salmonella-positive cantaloupes of 540 test cantaloupes by the rinse method. Overall, BPW, LAC, and UP broths were equivalent for the recovery of Salmonella from cantaloupes. Both the VIDAS ICS/SLM and TECRA Unique Salmonella methods detected significantly fewer Salmonella-positive cantaloupes than did the culture method: the VIDAS ICS/SLM method detected 23 of 50 Salmonella-positive cantaloupes (60 tested) and the TECRA Unique Salmonella method detected 16 of 29 Salmonella-positive cantaloupes (60 tested). The VIDAS SLM and culture methods were equivalent: both methods detected 37 of 37 Salmonella-positive cantaloupes (60 tested).

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.

AOAC International methods committee guidelines for validation of qualitative and quantitative food microbiological official methods of analysis.

Responding to a need for a guide for conducting Official Method validation studies of microbiological methods, AOAC utilized the experience of three microbiologists who have been active in the field of method validation. In collaboration, a document was prepared which covered the following areas: terms and their definitions associated with the Official Methods program (e.g., reference methods, alternative methods, and ruggedness testing), protocols and validation requirements for qualitative methods versus those for quantitative methods, the concept of the precollaborative study, ruggedness testing, tests for significant differences, performance indicators, and the approval process. After its preparation, this document was reviewed by the members of the Methods Committee on Microbiology and Extraneous Materials and by members of the Official Methods Board. Herein is presented the approved version of that document.

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.

Abbreviated Preenrichment Period for Recovery of Salmonella spp. from Selected Low-Moisture Dairy Foods.

A 6-h and a 24-h preenrichment procedure were compared for their ability to recover Salmonella spp. from selected low-moisture dairy foods. The foods were artificially inoculated several days before analysis, and 20 replicate test portions per procedure from each food were examined in each experiment. Samples examined by the 6-h abbreviated procedure were preenriched for 6 h at 35°C in an air incubator or water bath and centrifuged at 4,100 × g for 10 min. Pellets were suspended in tetrathionate broth and incubated for 24 h at 35°C. For the 24-h standard procedure, test portions were preenriched for 24 h at 35°C in an air incubator, subcultured to tetrathionate broth, and incubated for 24 h at 35°C. Selective enrichment broths from both procedures were streaked onto selective agar plates, and presumptive Salmonella isolates were identified by conventional biochemical and serological tests. Recovery of Salmonella spp. from instant nonfat dry milk and dry whole milk was equivalent for both preenrichment procedures. However, the relative effectiveness of the two procedures varied in the recovery of Salmonella spp. from noninstant nonfat dry milk, lactic casein, and rennet casein.

Comparison of Two Enzyme Immunoassays for Recovery of Salmonella spp. from Four Low-Moisture Foods.

Two enzyme immunoassays (Salmonella-Tek™ and Report™) were compared with the standard culture method of the Association of Official Analytical Chemists (AOAC) and the Food and Drug Administration's Bacteriological Analytical Manual (BAM) for the recovery of Salmonella spp. from four low-moisture foods. Two protocols were used to compare the effectiveness of the two immunoassays: i) foods were contaminated in the dry state; or ii) serial tenfold dilutions of Salmonella spp. were inoculated into the postenrichments after incubation. Of three hundred 25-g test portions inoculated in the dry state, 199 gave confirmed positive reactions with the Salmonella-Tek™ assay, 193 with the Report™ assay, and 206 with the AOAC/BAM method. There were seven false-negative reactions with Salmonella-Tek™ and 13 false negatives with the Report™, a false negative being defined as one that was negative by the enzyme immunoassay but was confirmed positive by the AOAC/BAM culture method. When the postenrichments were inoculated after incubation, a lower number of cells gave a positive assay result with the Salmonella-Tek™ system than with the Report™ system, indicating greater sensitivity.

Recovery of Salmonella from High-Moisture Foods by Abbreviated Selective Enrichment.

Five high-moisture foods were used to evaluate both the effect of a 6 h, rather than the standard 24 h, selective enrichment incubation period, and the efficiency of Rappaport-Vassiliadis (RV) medium relative to the use of selenite cystine (SC) and tetrathionate (TT) broths for the recovery of Salmonella . Cheese and lettuce were artificially inoculated with a pool of two serotypes, whereas the other foods were naturally contaminated. Significantly higher numbers of Salmonella -positive test portions were obtained at 24 h with the following food and media combinations: cheese (TT and RV media), lettuce (SC, TT, and RV media), raw chicken (RV medium), and pork sausage (SC, TT, and RV media). There were no significant differences between the two incubation periods in recovery of Salmonella from turkey. Overall, more Salmonella -positive test portions were obtained from samples of lettuce, chicken, and pork sausage selectively enriched in RV medium than in SC or TT broths. The results of this study indicate that not all high-moisture foods can be selectively enriched for 6 h without a significant loss in recovery of Salmonella . RV medium was superior to SC and TT broths for recovery of Salmonella from some meats and was at least as productive in its recovery from the other high-moisture foods tested.

Recovery of Salmonella from Frozen Shrimp: Evaluation of Short-Term Selective Enrichment, Selective Media, Postenrichment, and a Rapid Immunodiffusion Method.

Frozen shrimp was used as a high-moisture food matrix to evaluate the effect of the following conditions and media on the recovery of Salmonella : comparative efficiency of 6 and 24 h selective enrichment incubation periods; efficiency of Rappaport-Vassiliadis (RV) medium relative to selenite cystine (SC) and tetrathionate (TT) selective enrichment broths; need for postenrichment; and reliability of the immunodiffusion method ( Salmonella 1-2 TEST) as a rapid screening procedure. From a total of 244 Salmonella -positive, samples, recoveries at 6 h for selective enrichments SC, TT, RV(1) receiving 1 ml of inoculum, and RV(2) receiving 0.1 ml of inoculum, were 147, 149, 200, and 169, respectively; at 24 h, recoveries were 148, 142, 193, and 205, respectively. As a selective enrichment, RV medium was generally more productive than either SC or TT broths. Postenrichment reduced method sensitivity. Test kit reactions were read independently by three analysts to evaluate the immunodiffusion method. Examination of 200 shrimp samples by standard cultural and 1-2 TEST methods detected 52.5-57% and 56.5-60.5% positive samples, respectively.

The Survival of Shigella sonnei in Shredded Cabbage.

Commercially shredded cabbage distributed at the retail level is usually packaged under vacuum or in a modified atmosphere of nitrogen and carbon dioxide to suppress proliferation of aerobic spoilage microorganisms. The ability of Shigella sonnei to survive and grow in shredded cabbage packaged under these conditions was determined by the 3-tube most probable number procedure. After artificial inoculation with S. sonnei at one of three different levels, the shredded cabbage was packaged aerobically, under vacuum, or under modified atmosphere (30% nitrogen and 70% carbon dioxide) and stored at room temperature (24 ± 2°C) or under refrigeration (0-6°C). For most levels of inoculation, S. sonnei tended to increase or remain relatively high for 1-3 d in cabbage packaged under all three conditions and stored at room temperature. However, after 3 d of storage at this temperature, S. sonnei approached indeterminately low levels. The steady decrease of pH values of shredded cabbage over the storage period may have contributed to the decrease of S. sonnei . Under refrigeration, however, both the S. sonnei levels and the pH values remained relatively constant. The results indicate that S. sonnei can survive and even proliferate in shredded cabbage packaged and stored under a vacuum or modified atmosphere as well as aerobic conditions, thereby posing a potential hazard to the consumer.

Abbreviated Selective Enrichment, Post Enrichment and a Rapid Immunodiffusion Method for Recovery of Salmonella from Instant Nonfat Dry Milk.

Recovery of Salmonella from instant nonfat dry milk was studied by determining the efficiency of 6 and 24 h selective enrichment incubation periods, the productivity of Rappaport-Vassiliadis (RV) medium relative to selenite cystine and tetrathionate broths, and the potential enhancement of post enrichment with GN and M broths. Efficiency of the immunodiffusion method recently approved by the Association of Official Analytical Chemists (AOAC) for recovering Salmonella was also determined. Samples of artificially contaminated instant nonfat dry milk were selectively enriched for 6 h without compromising recovery. There was no advantage in using RV medium, and post enrichment did not enhance recovery. For the evaluation of the immunodiffusion method, three analysts independently read the test kit reactions. Of the 220 samples examined, 42.7-45.5% were positive by the immunodiffusion and the AOAC/Bacteriological Analytical Manual (BAM) culture methods, 42.3-43.2% were negative by both methods, 11.4-14.1% were negative by the immunodiffusion test and positive by the AOAC/BAM culture method, and 0-0.9% were negative by the AOAC/BAM method and positive by the immunodiffusion method.

Recovery of Salmonella from Fluid Milk.

Methodology was developed for isolation of Salmonella from skim milk, 2% fat milk, whole milk and buttermilk. Lactose broth, lactose broth plus brilliant green dye, buffered peptone water and each milk type plus brilliant green dye were evaluated as preenrichment broths. Incubation temperatures of 35 and 43°C were compared for use at the preenrichment stage. The recovery of Salmonella was determined after selective enrichment in selenite cystine, tetrathionate and Rappaport-Vassiliadis broths. Results indicated that fluid milk should be examined for Salmonella by being preenriched in lactose broth, subcultured to selenite cystine and tetrathionate broths and streaked to selective agars, with 35°C as the incubation temperature throughout the analysis.