Use of selective media to recover Salmonella and Vibrio cholerae after growth in reconditioned pork-processing wastewater.

Selective plating media are used for the enumeration and isolation of bacterial pathogens from food and water samples. This study compared the quantitative recovery of Salmonella spp. and Vibrio cholerae grown in nutrient-limited, filter-sterilized, reconditioned wastewater over the temperature range of 4 to 45 degrees C using nonselective and pathogen-specific selective media. Viable Salmonella were enumerated on tryptic soy agar (TSA) and XLT-4, and viable V. cholerae were enumerated on TSA and thiosulfate-citrate-bile-sucrose agar. There was a statistically significant (P < 0.05) higher recovery of both pathogens over the growth temperature range on TSA compared to the selective media. Trehalose, a stress-induced metabolite of Salmonella, was isolated from the cells grown in the reconditioned wastewater, whereas, the V. cholerae exhibited a change in cellular morphology from rod to coccoid shape. These results suggest that growth in nutrient-limited water injured or stressed the individual pathogens. Care should be used in choosing the procedure and plating medium for quantitative recovery of pathogens from such a nutrient-limiting environment.     

Validation of methods used to recover Escherichia coli O157:H7 and Salmonella spp. subjected to stress conditionst

Escherichia coli O157:H7, Salmonella spp., and Salmonella Typhimurium DT104 were stressed with lactic acid and cell-free supernatants from lactic acid bacteria and plated on three different media to determine if injured cells were recovered. A comparison of the susceptibility and recovery of antibiotic-resistant strains of the pathogens and nonresistant strains was also made. Acid stress conditions were created by adjusting the pH of a cocktail mixture (two to four strains) of the pathogen to 3.50 with lactic acid and holding for 18 h. The pathogen cocktail was also stressed with a cell-free supernatant of Lactobacillus lactis (pH 3.90) in a 4:6 ratio. Both nonstressed and stressed cocktail cultures were plated on Trypticase soy agar (TSA) and violet red bile agar (VRBA) for E. coli and xylose lysine tergitol4 (XLT4) for Salmonella. Repair of injured cells was evaluated by pour plating the stressed cells on a 5-ml thin layer of TSA and allowing a 2-h room temperature incubation followed by overlaying with VRBA or XLT4. There were significant reductions in the populations of both pathogens under both stress conditions when plating was done on nonselective media. Injured E. coli O157:H7 was not recovered on recovery or selective media compared with TSA. Numbers of cells of supernatant-stressed Salmonella spp. plated on selective and recovery media were similar to those on TSA. Acid-stressed cells for all Salmonella spp. were not recovered on TSA, selective, or recovery media at levels comparable to recovery on TSA. Antibiotic-resistant strains showed similar recovery patterns on all media evaluated. However, the antibiotic-resistant strains were less sensitive to both stress conditions. The use of antibiotic-resistant strains resulted in a greater recovery of stressed pathogens than the use of recovery media.     

Thin agar layer method for recovery of heat-injured Listeria monocytogenes

A thin agar layer (TAL) method was developed to recover heat-injured Listeria monocytogenes. Modified Oxford medium (MOX), a selective plating medium, inhibits heat-injured L. monocytogenes from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. In order to facilitate recovery of heat-injured L. monocytogenes cells while providing selectivity of isolation of L. monocytogenes from other bacteria in the sample, a unique TAL procedure was developed by overlaying 5 ml of nonselective medium (TSA) onto prepoured and solidified MOX medium in an 8.5-cm-diameter petri dish. The injured L. monocytogenes repaired and started to grow in the TSA during the first few hours after incubation of the plate. During the resuscitation of injured cells, the selective agents from MOX diffused to the TSA top layer to inhibit other microorganisms. L. monocytogenes showed a typical reaction (black colonies) on TAL after 24 h of incubation at 37 degrees C. The recovery rate for heat-injured L. monocytogenes with the TAL method was compared with those rates associated with TSA, MOX, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar after 3 h incubation). Milk and 0.1% peptone water that were inoculated with L. monocytogenes (4 to 5 log CFU/ml) were heated for 15 min at 55 degrees C. L. monocytogenes was enumerated on TSA, MOX, OV, and TAL media and procedures. No significant difference occurred among TSA, OV, and TAL (P > 0.05) in terms of enumeration of heat-injured L. monocytogenes, but these media recovered significantly higher numbers than did MOX agar (P < 0.05)-in both samples. The TAL method involves only one step, whereas OV is a more cumbersome two-step procedure.     

Inactivation of Salmonella typhimurium and Escherichia coli O157:H7 in apple juice by a combination of nisin and cinnamon

Pasteurized apple juice with nisin (0, 25, 50, 100, and 200 ppm, wt/vol) and cinnamon (0 and 0.3%, wt/vol) was inoculated with Salmonella Typhimurium and Escherichia coli O157:H7 at 10(4) CFU/ml and stored at 5 and 20 degrees C. Counts on tryptic soy agar (TSA), selective medium (xylose Lysine desoxycholate agar for Salmonella Typhimurium, and MacConkey sorbitol agar for E. coli O157:H7), and thin agar layer (TAL) were determined at 1 h and 1, 3, 7, and 14 days. The TAL method (selective medium overlaid with TSA) was used for recovery of sublethally injured cells. The pathogens were gradually inactivated by the acidic pH of apple juice. Nisin and cinnamon greatly contributed to the inactivation. The killing effect was more marked at 20 degrees C, with counts in all treated samples being undetectable by direct plating in 3 days for Salmonella Typhimurium and 7 days for E. coli O157:H7. Thus, several factors influenced the decrease in counts: low pH, addition of nisin and cinnamon, and storage temperature. The TAL method was as effective as TSA in recovering injured cells of the pathogens. The combination of nisin and cinnamon accelerates death of Salmonella Typhimurium and E. coli O157:H7 in apple juice and so enhances the safety of the product.     

Evaluation of direct plating methods to enumerate Alicyclobacillus in beverages

Ten agar media were evaluated for their suitability to support spore germination and colony development by six strains of Alicyclobacillus acidoterrestris, three strains of Alicyclobacillus acidocaldarius, and one strain of Alicyclobacillus cycloheptanicus. The influence of plating method (pour versus spread), incubation temperature (43 degrees C and 50 degrees C), and incubation time (up to 10 days) on colony development were determined. K agar, Alicyclobacillus medium (ALI agar), and Bacillus acidoterrestris thermophilic (BAT) agar recovered the highest numbers of spores. Orange serum agar and Hiraishi glucose yeast extract agar were the least suitable. Overall, surface plating was superior to pour plating and, with the exception of one strain of A. acidocaldarius which grew better at 50 degrees C, incubation of K agar, ALI agar, and BAT agar plates at 43 degrees C or 50 degrees C resulted in recovery of equivalent numbers of spores. Essentially all viable spores were detected on media incubated for 3 days at 43 degrees C. The ability of one strain of each Alicyclobacillus species to grow in ten non-carbonated commercially manufactured beverages at 30 degrees C and 43 degrees C was markedly affected by the composition of the beverages. Results show that surface plating samples on BAT agar, followed by incubating plates at 43 degrees C for 3 days provide the most suitable conditions to enumerate ten strains of three species of Alicyclobacillus most commonly responsible for spoilage of beverages.     

Media for detecting and enumerating yeasts and moulds.

Dilution plating techniques are designed to determine populations of viable fungal propagules per unit weight or volume of food. Direct plating techniques, on the other hand, are designed to assess the internal mycoflora of individual pieces of foods, e.g., seeds or dried fruits, and results are expressed as a percentage of infected pieces. Both techniques are used by industry and regulatory agencies to monitor levels of fungal contamination at various stages of food handling, storing, processing and marketing. Peptone (0.1%) water is commonly used as a diluent for samples to be homogenized or blended. Buffered diluents containing up to 30% glucose or 60% sucrose are recommended for enumerating xerophiles. No one medium is satisfactory for detection or enumeration of yeasts and moulds in all foods. Dichloran rose bengal chloramphenicol agar, oxytetracycline glucose yeast extract agar and rose bengal chloramphenicol agar are superior to acidified potato dextrose agar for enumeration of yeasts and moulds. Dichloran 18% glycerol agar performs well for enumerating moderately xerophilic yeasts and moulds. Fastidious xerophiles require media containing high concentrations of sugars and/or sodium chloride. Media have been formulated to detect potentially aflatoxigenic aspergilli and mycotoxigenic strains of penicillia and fusaria, but increased selectivity and specificity of media for detecting mycotoxigenic moulds are needed. Heat-resistant mould ascospores often require heat treatment prior to plating in order to activate the germination process. The spread-plate technique is strongly preferred over the pour-plate technique for enumerating yeasts and moulds. The recommended incubation temperature is 25 degrees C, but incubation time between plating and counting colonies ranges from 5 days for determination of general populations of mycoflora to 4 weeks or more for fastidious xerophiles. There is a need for new and improved media for selectively isolating various groups, genera, species and/or strains of fungi capable of growing only under specific environmental conditions, e.g., low aw or, in the case of sublethally injured cells, under conditions which facilitate resuscitation. Improved media are needed which accurately detect moulds producing specific mycotoxins in a wide range of food types.     

Application of thin agar layer method for recovery of injured Salmonella typhimurium

Xylose lysine decarboxylase (XLD) medium, a selective plating medium, can inhibit heat-injured Salmonella typhimurium from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. To facilitate recovery of heat-injured S. typhimurium cells while providing selectivity of isolation of S. typhimurium from other bacteria in the sample, a thin agar layer (TAL) procedure was developed by overlaying 14 ml of nonselective medium (TSA) onto prepoured and solidified XLD medium in a 8.5 cm diameter Petri dish. During the first few hours of incubating the plate, the injured S. typhimurium repaired and started to grow in the TSA. During the resuscitation of injured cells, the selective agents from XLD were diffused to the TSA top layer part. Once the selective agents diffused to the top part of the TAL, the resuscitated S. typhimurium started to produce a typical reaction (black color) and other microorganisms were inhibited by the selective agents. The recovery rate for heat-injured (55 degrees C for 15 min) S. typhimurium with the TAL method was compared with TSA, XLD, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar 3-4 h after incubation). No significant difference occurred among TSA, OV, and TAL (P > 0.05) for enumeration of heat-injured S. typhimurium, but they recovered significantly higher numbers than from XLD agar (P < 0.05).     

Evaluation of universal preenrichment broth for growth of heat-injured pathogens.

Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen. Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined. Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Enteritidis. and Listeria monocytogenes in UPB. Bacterial cells were heat injured in tryptic phosphate broth at 57.2 degrees C and inoculated at populations of ca. 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples. Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37 degrees C postinoculation, and pathogens were enumerated on appropriate selective media. Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (> 10(3) CFU/ ml) for detection by most immunoassays or PCR assays. Cells often required 3 to 4 h for recovery before growth was initiated. Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml. At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml. Enrichment in UPB at 37 degrees C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes reliably provides at 24 h of incubation-but not at 6 h-sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml. These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.     

Evaluation of thin agar layer method for recovery of acid-injured foodborne pathogens.

The thin agar layer (TAL) method of Kang and Fung was used to enumerate acid-injured foodborne pathogens. This method involves overlaying 14 ml of nonselective medium (tryptic soy agar [TSA]) onto a prepoured and solidified pathogen-specific, selective medium in a petri dish. After surface plating, injured cells resuscitated and grew on TSA during the first few hours of incubation; then, the selective agents from the selective medium diffused to the top layer, interacted with the recovered microorganisms, and started to produce typical reactions. Foodborne pathogens were exposed to 2% acetic acid for 1, 2, or 4 min, and the recovery rate with the TAL method was compared with the rate of TSA and pathogen-specific, selective media. No significant difference occurred between TSA and TAL (P > 0.05) for enumeration of acid-injured Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, and Yersinia enterocolitica, and both recovered significantly higher numbers than the selective medium for each respective pathogen (P < 0.05). For recovery of acid-injured Listeria monocytogenes, no difference (P > 0.05) occurred among TSA, TAL, and selective media. However, fewer cells were recovered in the selective media. The TAL method is a one-step, convenient procedure for recovery of acid-injured cells.     

Efficacy of prolonged (48 h) selective enrichment for the detection of foodborne Salmonella.

The effect of prolonged (48 h) incubation on the productivity of five enrichment-temperature conditions (tetrathionate brilliant green, 35 and 43 degrees C; Muller-Kauffman tetrathionate brilliant green, 43 degrees C; Rappaport-Vassiliadis, 43 degrees C; selenite cystine, 35 degrees C) was compared to homologous results obtained under standard (24 h) conditions of selective enrichment. Of 797 high moisture and 166 low moisture foods tested, 171 (21.5%) and 80 (48.2%), respectively, were found to contain salmonellae by one or more analytical condition. Combined results of the five enrichment conditions after 24 and 48 h of incubation identified 247 (98.4%) and 250 (99.6%) of the 251 contaminated samples identified in this study. Our results are at variance with earlier reports on the greater method sensitivity with extended (greater than or equal to 48 h) periods of selective enrichment. The productivities of individual enrichment conditions after each period of incubation varied markedly where recovery rates with TBG43 and MKTBG43 exceeded that obtained with SC35 and TBG35. Our findings also underline the determinant role of enrichment at an elevated temperature (43 degrees C), and use of multiple enrichment and plating media for the optimal recovery of foodborne Salmonella.     

Detection of low numbers of healthy and sub-lethally injured Salmonella enterica in chocolate

The capacity to detect low levels of healthy and sub-lethally injured Salmonella enterica cells in chocolate by two alternative rapid detection methods iQ-Check(TM)Salmonella II real-time PCR (Bio-Rad) and VIDAS? Easy SLM (BioMérieux) was assessed and compared with ISO 6579:2005. Chocolate, a low moisture food known to support the survival of Salmonella, was challenged as food matrix. Buffered peptone water (BPW) did not support the recovery of low levels of sub-lethally injured S. enterica independent of the detection method, while BPW supplemented with milk powder enabled detection by the three examined methods. However, inhibition of real-time PCR was observed since for one out of three repetitions of chocolate inoculated with a low number of sub-lethally injured S. enterica cells, no PCR signal was obtained. Therefore, attention should be paid to the enrichment step to avoid false negative results due to the presence of especially sub-lethally injured Salmonella cells in chocolate. An appropriate sample preparation (such as enrichment media and conditions for incubation) remains the key factor for reliable detection including sub-lethally injured cells and should be evaluated, if necessary optimized, for each detection assay.     

Evaluation of selective plating media for the detection of heat- or freeze-injured Staphylococcus aureus

The efficacy of Baird-Parker (BP) agar, mannitol-salt-egg yolk (MSEY) agar and mannitol salt (MS) agar in detecting Staphylococcus aureus FRI-100 heated at 52 degrees C for 20 min in 100 mmol/L potassium phosphate buffer was determined. Brain heart infusion agar with 1% pyruvate (BHIP agar) supported the highest recovery of injured cells and was used as the control medium. Of the three selective media, significantly higher recovery of heat-injured cells was observed on BP agar than MSEY agar, and the poorest recovery was observed on MS agar (p < 0.05). Low recovery of unheated cells was obtained for MS compared with other media (p < 0.05). A reduction in populations occurred gradually in reagent-grade water stored for 14 days at -20 degrees C. There was no significant difference between BHIP agar and MS agar in the number of freeze-injured cells recovered from 1 to 14 days.     

Thermal resistance of heat-, cold-, and starvation-injured Salmonella in irradiated comminuted Turkey

To investigate the effects of sublethal stress on Salmonella thermal inactivation kinetics, an eight-strain Salmonella cocktail was subjected to heat shock (30 min at 54 degrees C), cold shock (2 h at 4 degrees C), and starvation stress (10 days in phosphate buffer at 4 degrees C), harvested by centrifugation, and inoculated into irradiated comminuted turkey. Immediately after stressing, the Salmonella cocktails contained 89.1% heat-injured, 44.7% cold-injured, and 67.7% starvation-injured cells, as determined by plating on selective and nonselective media. D60 degrees C-values for the heat-shocked cocktail (0.64 min on Trypticase soy agar containing 0.6% yeast extract [TSAYE], 0.35 min on xylose lysine desoxycholate [XLD] agar) were higher (P < 0.05) than those for the unshocked control (0.41 min on TSAYE, 0.17 min on XLD), whereas D60 degrees -values for the cold-shocked cocktail (0.38 min on TSAYE, 0.17 min on XLD) were not significantly different from those for the control. Starved cells had the same D60 degrees C-value on TSAYE as did the unshocked cocktail, but the D60 degrees C-value on XLD was significantly lower (0.14 min). Although starvation and cold shock were not thermally protective, heat shock increased thermal resistance, indicating that product history and the physiological state of the Salmonella cells should be considered when developing and validating thermal processes. D60 degrees C-values observed on selective media were significantly lower than those observed on nonselective media for all stress treatments and for the control. Therefore, nonselective culture media should be used to assess the response of microorganisms to a thermal challenge when developing performance standards for lethality.     

A comparative study between overlay method and selective-differential media for recovery of stressed Enterobacter sakazakii cells from infant formula

This study compares the performance of different selective-differential media with the overlay method for recovery of stressed cells of Enterobacter sakazakii from infant formula milk (IFM). Five different selective-differential media were used in this study: OK medium, violet red bile agar (VRBA), Druggan-Forsythe-Iversen agar (DFI), Enterobacteriaceae enrichment (EE) agar, and fecal coliform agar (FCA). Tryptic soy agar supplemented with 0.1% sodium pyruvate (TSAP) was used as a control. The overlay method involved applying a thin layer (8ml) of each of the selective media onto TSAP after spreading a sample onto TSAP. Reconstituted IFM was inoculated by ca 1x10(7)CFU/ml of a mixture of four strains of E. sakazakii and subjected to different stress conditions: heat (55 degrees C for 10min), a freeze-thaw cycle (-20 degrees C for 24h, thawed at room temperature, frozen again at -20 degrees C, and thawed), acidic pH (pH 3.56 for 15min), alkaline pH (pH 11.04 for 15min), and desiccation (E. sakazakii was inoculated onto powdered IFM at a level of ca 1x10(6)CFU/g, held at 21 degrees C, water activity of the inoculated product was 0.29 and examined at 0, 15, and 30d). No major differences were noticed between the control (TSAP) and the overlay methods. However, the overlay method recovered significantly higher numbers of stressed E. sakazakii cells compared to selective-differential media. Also, the selective-differential media exhibited some variability in terms of their capabilities to recover stressed cells of E. sakazakii. Among all the examined selective-differential media, DFI performed better for recovering stressed E. sakazakii cells. This study suggests that the overlay method may serve as a potential alternative to direct selective plating for best recovery of E. sakazakii from IFM.     

Survival and recovery of viable but nonculturable Listeria monocytogenes cells in a nutritionally depleted medium

Survival of a desiccated five-strain Listeria monocytogenes mixture during storage in sand at 4 degrees C for 2 months was determined using the acridine orange direct count method with novobiocin and plate counts. Samples of inoculated sand were taken every 2 weeks, incubated at 37 degrees C for 6 h, stained with acridine orange, and then examined with a fluorescence microscope. Elongated viable but nonculturable cells were most frequently observed during weeks 2 and 4. At weeks 6 and 8, most of the cells either remained viable or were dead. In each microscopic field, only one or two viable but nonculturable cells were observed among hundreds of other viable culturable cells, indicating that L. monocytogenes does not generally become viable but nonculturable. Therefore, viable but nonculturable cells are not a concern when plating environmental samples or desiccated L. monocytogenes cells on nonselective media. Tryptic soy agar with 0.6% (wt/vol) yeast extract (TSAYE) and Columbia agar were used as nonselective plate count media. Modified Oxford agar and TSAYE + 5% (wt/vol) sodium chloride were used as the selective plate count media. The effects of aerobic or anaerobic incubation and media supplementation with 0.1% or 1% (wt/vol) sodium pyruvate were tested to optimize recovery of desiccated cells. Nonselective media showed better recovery when TSAYE and Columbia agar contained 0.1% (wt/vol) pyruvate and were incubated aerobically. These two culture methods were equally effective (P > 0.05) for recovering desiccated L. monocytogenes cells.     

Fate of Escherichia coli O157:H7 and Listeria monocytogenes in strawberry juice and acidified media at different pH values and temperatures

Survival and growth of Escherichia coli O157:H7 and Listeria monocytogenes in strawberry juice and acidified media at different pH levels (pH 3.4 to 6.8) and temperatures were studied. Sterile strawberry juice (pH 3.6) and acidified trypticase soy broth (TSB) media (pH 3.4 to 6.8) were inoculated with approximately 6.7 log CFU/ml E. coli O157:H7 or 7.3 log CFU/ ml L. monocytogenes, incubated for 3 days at 4 and 37 degrees C. Bacterial levels were determined after 2 h, 1 day, and 3 days using surface plating nonselectively on tryptic soy agar and selectively on sorbitol MacConkey agar for E. coli O157:H7 or modified Oxford agar for L. monocytogenes. A spectrophotometer (660 nm) was also used to study growth inhibition of L. monocytogenes in different TSB and strawberry juice media (pH 3.4 to 7.3). E. coli O157:H7 survived well at pH values of 3.4 to 6.8 at 4 degrees C, but the number of injured cells increased as pH decreased and incubation time increased. At 37 degrees C, E. coli O157:H7 was inactivated at pH of < or = 3.6 but could grow at pH 4.7. L. monocytogenes was quickly injured at pH of < or = 4.7 within 2 h of storage at 4 degrees C and then was slightly and gradually inactivated as storage time increased. L. monocytogenes survived well at pH 6.8 at 4 degrees C and grew well at 37 degrees C. Growth of L. monocytogenes at 37 degrees C was inhibited in TSB by 1% citric acid and 0.5% malic acids at pH 3.4 or by 50% strawberry juice at pH 4.7. Bacterial injury and inactivation appeared to be induced by the acids in strawberry juice. The acids, pH value, temperature, and time were important factors for bacterial survival, inactivation, and growth in the media tested.     

Comparison of recovery methods for freeze-injured Listeria monocytogenes,Salmonella Typhimurium,and Campylobacter coli in cell suspensions and associated with pork surfaces

Cells injured as a result of freezing, heating, and acidification treatments may not grow during conventional microbiological procedures owing to the presence of selective agents, compounds, or dyes in the media, impairing the cell's ability to repair itself and grow. Injured cells can be recovered by combining selective and nonselective media into a single system. With such combinations, the diffusion of the selective compounds or dyes is controlled, allowing for the resuscitation of injured cells of interest while also inhibiting the growth of undesirable background microflora. In this study, Listeria monocytogenes, Salmonella Typhimurium, and Campylobacter coli suspended in buffer or associated with pork surfaces were subjected to a freeze-thaw cycle (-15 degrees C for 24 h, 4 degrees C for 4 h). Following treatments, freeze-injured cells were plated on appropriate media for the overlay (OV), thin agar layer (TAL), and Lutri plate (LP) recovery methods. The levels of L. monocytogenes and Salmonella Typhimurium recovered from cell suspensions and pork surfaces by the TAL, OV, and LP methods following freeze treatments were not statistically different (P > 0.05) from recovery levels associated with nonselective media. Conversely, levels of pathogens on selective media were significantly reduced compared with those for the other methods employed. The TAL method's recovery of C. coli was not significantly different from that achieved with the nonselective media. Overall, the results presented in this study demonstrate that the TAL method not only was easier to perform, but also allowed improved isolation of single colonies for further characterization. This study may provide researchers with better methods to determine the effectiveness of industry-employed chilling processes in reducing pathogenic bacteria associated with red meat surfaces.     

Recovery of Listeria monocytogenes on selective agar media in a collaborative study using reference samples

Sixteen laboratories compared counts of Listeria monocytogenes in reference samples using Blood agar, Palcam(y) agar and Oxford agar. Significant differences were found between laboratories. The mean counts on Blood agar were significantly higher than on Palcam(y) or Oxford agar. The mean counts on Palcamy agar were somewhat higher than on Oxford agar (only after 48 h incubation), but no significant difference was found. Addition of egg yolk to Palcam agar seems to be beneficial for the recovery of sublethally injured cells. Recovery of L. monocytogenes was higher after 48 h incubation for all media tested.     

Development of methods for the recovery of Escherichia coil O157:H7 and Salmonella from beef carcass sponge samples and bovine fecal and hide samples

Culture methods were developed for the concurrent recovery of Escherichia coli O157:H7 and Salmonella from bovine carcass, hide, and fecal samples. Several enrichment conditions were tested for the overall growth of pure cultures; tryptic soy broth for 2 h at 25 degrees C and then for 6 h at 42 degrees C was the protocol selected for use. Immunomagnetic separation (IMS) was incorporated for sensitivity and selectivity, along with a post-IMS enrichment for the recovery of Salmonella as recommended by the manufacturer. Selective agars for plating after IMS were chosen on the basis of ease of target colony identification. Sorbitol MacConkey agar supplemented with cefixime and potassium tellurite and Rainbow agar supplemented with novobiocin and potassium tellurite were chosen for the recovery of E. coli O157:H7. Brilliant green agar with sulfadiazine and Hektoen enteric agar supplemented with novobiocin were selected for the recovery of Salmonella. The resulting methods were evaluated along with standard or previously used methods for the recovery of E. coli O157:H7 and Salmonella from bovine hide and fecal samples and carcass sponge samples. The Meats Research Unit (MRU) methods performed at least as well as the established methods, except that a secondary enrichment in tetrathionate (TT) broth prior to IMS was required for the optimal recovery of Salmonella from feces. Thus, the MRU and MRU-TT methods are effective in the recovery of both E. coli O157:H7 and Salmonella from a single bovine carcass, hide, or fecal sample.