Wallace H. Andrews

Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium for the recovery of Salmonella spp. from foods with a low microbial load

The relative effectiveness of Rappaport-Vassiliadis (RV) medium, selenite cystine (SC) broth, and tetrathionate (TT) broth for the recovery of Salmonella spp. from foods with a low microbial load was determined. RV medium made from its individual ingredients and incubated at 42 degrees C was compared with a commercial preparation of SC broth, incubated at 35 degrees C, and TT broth incubated at 35 and 43 degrees C, for the recovery of Salmonella spp. Twenty-one artificially contaminated food types that included dairy foods, spices, and egg products, as well as other low-microbial-load foods, were analyzed. The foods were inoculated with single Salmonella serovars at target levels ranging from 0.04 to 0.4 CFU/g. No significant differences (P< or =0.05) among the selective enrichment broths for the recovery of Salmonella spp. from 18 of the foods were observed. Significantly fewer Salmonella-positive test portions of gelatin, guar gum, and nonfat dry milk were recovered with RV medium than with SC broth incubated at 35 degrees C and TT broth incubated at 35 and 43 degrees C. TT broth incubated at 35 degrees C recovered the greatest number of Salmonella-positive test portions. For the recovery of Salmonella spp. from foods with a low microbial load, it is recommended that TT broth incubated at 35 degrees C and RV medium incubated at 42 degrees C be used.

Resuscitation of Injured Salmonella spp. and Coliforms from Foods

Because damaged cells may account for a substantial proportion of the bacterial population in processed foods, the food microbiologist must choose the most appropriate methods for detecting damaged as well as noninjured cells. Any method intended to recover damaged organisms should include a resuscitative, or repair, process that will restore the injured cells to a sound physiological condition before subjecting them to the severity of selective enrichment media. It should also provide a reliable indication of the microbiological safety and quality of any particular food. This paper reviews various factors that affect the recovery of Salmonella spp., which include: (a) sample rehydration, (b) period of preenrichment, (c) incubation in both aerobic and anaerobic environments, (d) media composition and (e) the relative merits of preenrichment and direct selective enrichment. Because resuscitation of injured Salmonella cells does not occur during the selective enrichment step and beyond, the effect and interaction of these factors are considered primarily for the preenrichment step of the isolation procedure for Salmonella . This paper also reviews five methods recently developed for recovery of coliforms, which include: (a) hydrophobic grid membrane filtration, (b) radiometry, (c) electrical impedance, (d) fluorogenic assay and (e) the Petrifilm system. Each of these methods may incorporate a step for resuscitation of injured organisms.

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.

Preenrichment versus direct selective agar plating for the detection of Salmonella Enteritidis in shell eggs

The relative effectiveness of two methods for the recovery of Salmonella Enteritidis (SE) from jumbo and medium shell eggs was compared. The first method used in the comparison consisted of a preenrichment of the sample, and the second method was developed by the U.S. Department of Agricultures Animal and Plant Health Inspection Service (APHIS). Three bulk lots of blended, pooled eggs, each containing 220 liquid whole eggs that were thoroughly mixed manually were artificially inoculated with different levels of SE cells between approximately 10(0) and 10(3) CFU/ml. Twenty samples containing the contents of approximately 10 eggs each (by weight) were withdrawn from each of the inoculated bulk lots and incubated for 4 days at room temperature (ca. 23 degrees C). For the APHIS method, each sample was cultured by direct plating onto brilliant green (BG), brilliant green with novobiocin (BGN), xylose lysine desoxycholate (XLD), and xylose lysine agar Tergitol 4 (XLT4) agars. For the preenrichment method, 25-g portions from each pool were enriched in modified tryptic soy broth with 30 mg/liter of FeSO4. After 24 h of incubation, the preenrichments were subcultured to tetrathionate and Rappaport-Vassiliadis broths, and streaked to BG, BGN, bismuth sulfite, XLD, and XLT4 agar plates. SE isolates were confirmed biochemically and serologically. In all of the experiments, the preenrichment method recovered significantly more SE isolates (P < 0.05) of all the phage types and inoculum levels than did the APHIS method. From a total of 539 jumbo egg test portions analyzed, 381 (71%) were SE-positive by the preenrichment method and 232 (43%) were positive by the APHIS method. From a total of 360 medium egg test portions analyzed, 223 (62%) were SE-positive by the preenrichment method and 174 (48%) were positive by the APHIS method. The preenrichment method provided greater sensitivity for the isolation of SE in contaminated egg slurries than did the APHIS method.

An improved method for the recovery of Salmonella serovars from orange juice using universal preenrichment broth.

The relative effectiveness of three methods for the recovery of Salmonella serovars from orange juice was determined. One method, a modified Bacteriological Analytical Manual (BAM) procedure consisted of preenrichment in lactose broth at 35 degrees C for 24 h, selective enrichment, and selective plating. Another method, a National Centers for Disease Control and Prevention (CDC 1) procedure, consisted of direct enrichment in tetrathionate broth at 35 degrees C for 24 and 48 h, followed by selective plating. The third method (also from CDC and designated CDC 2) consisted of preenrichment in Universal Preenrichment (UP) broth at 35 degrees C for 24 h, selective enrichment, and selective plating. In 10 experiments encompassing five different Salmonella serovars and 200 test portions per broth, the CDC 1 method recovered 141 Salmonella-positive test portions, the BAM method recovered 151, and the CDC 2 method recovered 171. In 2 of the 10 experiments, with two different Salmonella serovars, the BAM recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did the CDC 2 method. On the basis of the above results, the second phase of this study focused on a comparison of the effectiveness of the BAM-recommended lactose broth and the CDC 2-recommended UP broth as preenrichment media for the recovery of Salmonella serovars from pasteurized and unpasteurized orange juice. Subsequent culture treatment of the two preenrichments was identical so that the effect of other variables (e.g., different selective enrichment media, various incubation temperatures, and different selective plating agars) on the relative performance of these two preenrichment media was excluded. In one of nine experiments, with pasteurized orange juice, lactose broth recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did UP broth. For the combined results of the nine pasteurized orange juice experiments (180 test portions per broth), lactose broth recovered 99 Salmonella-positive test portions, and UP broth recovered 116. In three of seven experiments, with unpasteurized orange juice, lactose broth recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did UP broth. For the combined results of the seven unpasteurized orange juice experiments (140 test portions per broth), lactose broth recovered 73 Salmonella-positive test portions, and UP broth recovered 117. For both pasteurized and unpasteurized orange juice, the total number of Salmonella-positive test portions recovered with UP broth was significantly greater than the number recovered with lactose broth. These results indicate that UP broth is a more effective enrichment broth for the recovery of Salmonella from orange juice than is lactose broth.

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).

Potential Role of Refrigerated Milk Packaging in the Transmission of Listeriosis and Salmonellosis

Cultures of three Listeria monocytogenes serotypes and three Salmonella spp. were applied to the exterior surfaces of waxed cardboard or plastic milk containers. Contamination sites were sampled with premoistened cotton swabs during 14 d of refrigeration. Unstressed cells of Listeria survived up to 14 d on the surfaces of waxed (1 serotype) and plastic (3 serotypes) containers. Heat-stressed cells of all three serotypes of Listeria survived for 2 d on both types of containers. One serotype survived for 4 d, but only on plastic containers. Unstressed cells of all three Salmonella strains survived up to 14 d on both types of containers. Heat-stressed Salmonella strains survived up to 2 d (waxed containers) and 4 d (plastic containers).

Validation of modern methods in food microbiology by AOAC INTERNATIONAL collaborative study

Abstract There is a common misperception that the validation of a microbiological method by an AOAC collaborative study is an arduous undertaking. This paper attempts to dispel that notion by explaining how the system works, identifying the key players, and delineating their responsibilities. Criteria useful in selecting a procedure to be studied collaboratively and the clearance procedures for proposed study protocols and reports of completed studies are discussed. The author offers his views on the evolving internationality of AOAC and suggests that the harmonization of method validation by AOAC and other international organizations would be to the benefit of all.

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 Administrations 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.

Sulfite Compounds as Neutralizers of Spice Toxicity for Salmonella

The ability of five inorganic chemical salts (K2SO3, K2SO4, Na2SO3, Na2SO4, and CaCO3) to neutralize the toxicity of six spices (allspice, cinnamon, clove, garlic powder, onion powder, and oregano) for Salmonella was evaluated. Their effect on four spices non-toxic to Salmonella (black pepper, white pepper, rosemary, and thyme) was also determined. The inhibitory effects of onion and garlic powders were overcome by addition of 0.5% of K2SO3 or Na2SO3 to pre-enrichments of lactose broth, nutrient broth, or trypticase soy broth. Allspice, cinnamon, clove, and oregano remained toxic to Salmonella in all pre-enrichment broths tested, with or without chemical additives. None of the chemical additives had any effect upon isolation of Salmonella from the non-toxic spices. Until a more practical method for analysis of allspice, cinnamon, clove, and oregano is developed, dilution of these spices to non-toxic levels is recommended.