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.     

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.     

Comparison of recovery of airborne microorganisms in a dairy cattle facility using selective agar and thin agar layer resuscitation media

Thin agar layer (TAL) medium was developed at Kansas State University to improve the resuscitation of injured cells and has been shown to result in higher recovery than is obtained with selective media alone for cold-, heat-, salt-, and acid-injured cells. The experiment presented here was designed to determine the effectiveness of the TAL method for the recovery of possibly injured organisms from air. Eleven agar media were used for the experiment: tryptic soy agar (TSA), MacConkey sorbitol agar (MSA), TAL-MSA, Baird-Parker (BP) agar, TAL-BP agar, modified Oxford (MOX) agar, TAL-MOX agar, xylose lysine sodium desoxycholate (XLD) agar, TAL-XLD agar, Yersinia-selective (CIN) agar, and TAL-CIN agar. The TAL plates were prepared by pipetting 6 ml of selective agar into a BBL Rodac plate (65 by 15 mm). Selective agar was allowed to solidify, and then each plate was overlaid with 6 ml of TSA. Selective agar plates were prepared by pipetting 12 ml of agar into BBL Rodac plates and allowing the agar to solidify. Samples were taken at an indoor cattle facility at five separate locations with a BioScience SAS air-sampling instrument. For each plate, 60 liters of air was sampled. Three replications of the experiment were performed. The TAL method resulted in higher counts of microorganisms on all media tested. In addition, 175 isolates were selected randomly and identified in order to test the selectivity of TAL and the selective media for target organisms. The data obtained in this study show that the TAL resuscitation method is effective and necessary for the recovery of airborne organisms that may be injured.     

SIMULTANEOUS RECOVERY AND DETECTION OF FOUR HEAT-INJURED FOODBORNE PATHOGENS IN GROUND BEEF AND MILK BY A FOUR-COMPARTMENT THIN AGAR LAYER PLATE

A four‐compartment thin agar layer (4‐TAL) system was developed to improve operation efficiency and recover injured foodborne pathogens simultaneously. The system consisted of a layer of nonselective agar overlaid on four different selective agars (xylose lysine desoxycholate [XLD], cefsulodin irgasan novobiocin [CIN], modified Oxford medium [MOX] and MacConkey sorbitol agar [MSA]) housed in a four‐compartment petri dish. We applied this system to simultaneously recover heat‐injured (55C, 10 min) Escherichia coli O157:H7 (MSA), Listeria monocytogenes (MOX), Salmonella Typhimurium (XLD) and Yersinia enterocolitica (CIN) from ground beef and pasteurized milk. No significant difference (P > 0.05) occurred between the single recovery unit (nonselective agar overlaid on one selective agar in a standard petri dish) and the 4‐TAL for detecting four heat‐injured pathogens in tested samples. Both TAL methods showed greater recovery of four heat‐injured pathogens than the pathogen‐specific selective media (P < 0.05). The 4‐TAL system appears to be efficient for recovery and detection of injured pathogens in food in terms of operation, material and labor costs, and space of incubation.     

Efficacy of the thin agar layer method for the recovery of stressed Cronobacter spp. (Enterobacter sakazakii)

Cronobacter spp. (Enterobacter sakazakii) are emerging opportunistic pathogens for all age groups, and are of particular concern when it comes to infants. Prior to contaminating food, the organism may be exposed to a variety of stresses, leading to a generation of sublethally injured cells that may not be detected by selective media unless a protracted recovery period is included in the isolation procedure. This study evaluated the efficacy of the thin agar layer (TAL) method for the recovery of Cronobacter cells that had been exposed to various stress conditions. Five strains of C. sakazakii and C. muytjensii were exposed to starvation, heat, cold, acid, alkaline, chlorine, or ethanol, with or without further exposure to desiccation stress. The recovery of the stressed cells was determined on tryptone soy agar (TSA; nonselective control medium), violet red bile glucose agar (VRBGA; selective agar), Druggan-Forsythe-Iversen (DFI; selective agar), and TAL media (viz., VRBGA overlaid with TSA, and DFI overlaid with TSA). Regardless of stress type, there were no significant differences among the recoveries of stressed desiccated Cronobacter spp. cultures on TSA, DFI+TSA, and VRBGA+TSA, but there was significantly less recovery on VRBGA. The recovery of prestressed desiccated Cronobacter spp. on DFI+TSA was similar to that on TSA, whereas the recovery on VRBGA+TSA was lower. DFI+TSA performed better than VRBGA+TSA did in differentiating Cronobacter spp. within mixed bacterial cultures. The results of this study suggest the use of the TAL method DFI+TSA as an improved method for the direct recovery of stressed Cronobacter spp.     

Simultaneous Recovery of Four Heat-injured Foodborne Pathogens from a Four-compartment Thin Agar Layer Plate

ABSTRACT: We have developed and tested a 1-step resuscitation thin agar layer (TAL) system (nonselective agar overlaid on selective agar) to effectively recover heat-, acid-, cold-, and salt-injured foodborne pathogens in liquid foods. Each TAL unit was designed for the recovery of one injured population. To improve operation efficiency we have developed a single 4-compartment Petri dish to simultaneously recover heat-injured Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium , and Yersinia enterocolitica. No significant difference occurred between the single recovery unit and the 4-compartment unit for enumeration of 4 heat-injured pathogens (P > 0.05). This 4-compartment TAL system is at least 4 times more efficient than the single TAL system.     

Evaluation of Thin Agar Layer Method for Recovery of Heat-Injured Foodborne Pathogens

ABSTRACT The Thin Agar Layer (TAL) method of Kang and Fung 1998 was used to enumerate heat‐injured (55 °C for 10 min) foodborne pathogens. This method involves overlaying 14 mL of nonselective medium (Tryptic Soy Agar, TSA) onto a prepoured, pathogen‐specific, selective medium. The recovery rate with the TAL method was compared with the rate for TSA and pathogen‐specific, selective media. No significant differences occurred between TSA and TAL (P > 0.05) for enumeration of heat‐injured Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Yersinia enterocolitica, and both recovered significantly higher numbers than selective media (P < 0.05). The TAL method is a 1‐step, convenient procedure for recovery of heat‐injured cells.     

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.     

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.     

A solid agar overlay method for recovery of heat-injured Listeria monocytogenes

A solid agar overlay method was developed for recovery of heat-injured Listeria monocytogenes. Presolidified nonselective tryptic soy agar with 0.6% yeast extract (TSAYE, 2% agar) was overlaid on top of solidified modified Oxford agar (MOX). Heat injury of L. monocytogenes was conducted at 58 degrees C for 6 min in a jacketed flask filled with tryptic soy broth. Both noninjured and heat-treated L. monocytogenes cells were plated onto TSAYE, MOX, and TSAYE-MOX plates. No significant differences (P > 0.05) in recovery were found among the three media for noninjured bacterial cells. Recovery of heat-injured L. monocytogenes cells on TSAYE-MOX overlay plates was equivalent to that on the nonselective TSAYE medium, whereas recovery on the selective MOX medium was significantly lower (P < 0.05) compared with both TSAYE and the overlay plates. There were no significant differences (P > 0.05) among the overlay plates prepared 0, 2, 4, 6, 8, 16, and 24 h prior to plating heat-injured bacterial cells. The TSAYE-MOX overlay also allowed differentiation of L. monocytogenes from a mixture of four other types of foodborne pathogens. This solid agar overlay method for recovery of heat-injured L. monocytogenes cells is less time-consuming and less complicated than the conventional overlay-underlay technique and the double overlay modification of the thin agar layer method and may allow for greater laboratory plating efficiencies.     

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.     

Recovery of heat-injured Listeria innocua

Listeria innocua was subjected to thermal inactivation and the extent of heat-injured cells was quantified. Cultures were heated in liquid medium for different times, using temperatures in the range of 52.5 to 65.0 degrees C, and plated on Tryptic Soy Agar with 0.6% yeast extract (TSAYE) used as non-selective medium and on TSAYE plus 5% NaCl (TSAYE+NaCl) and Palcam agar with selective supplement (Palcam agar) as selective media. The difference observed in counts in non-selective and in selective media gave an indication of cell injury during the heat treatment. D- and z- values were calculated for all conditions considered. For each temperature, D-values obtained using non-selective recovery procedures were higher than the ones obtained using the two selective media. When comparing the selective media, it can be concluded that Palcam agar allowed recovery and growth of thermally injured cells and so it was less inhibitor than TSAYE+NaCl. Another important result was the influence of temperature on the degree of cellular injury. As temperature increases, the degree of heat-injured cells also increases, and consequently concern has to be taken with the temperature and the counting medium used in food processing studies. The results of this work clearly demonstrated that selective media used for Listeria monocytogenes enumeration/detection might not be suitable for the recovery of heat-injured cells, which can dangerously underestimate the presence of this foodborne pathogen.     

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.     

Combination of Carbon Dioxide and Cinnamon to Inactivate Escherichia coli O157:H7 in Apple Juice

ABSTRACT: Pasteurized apple juice with CO2 (0, 1, and 4%) and cinnamon (0 and 0.3%) was inoculated with Escherichia coli O157:H7 at 104 CFU/mL, and stored at 5 and 20 °C. Counts on nonselective and selective media, and thin agar layer (TAL; selective medium overlaid with nonselective medium) were determined at 1 h and 1, 3, 7, and 14 d. Inactivation was greater at 20 °C. Samples with 1 and 4% CO₂, alone and combined with cinnamon, presented < 0.7 log CFU/mL in 3 d. Counts in apple juice inoculated at 10² CFU/mL, a low-level E. coli O157:H7 contamination, were nondetectable at 3 d. The TAL method was as effective as nonselective medium to recover injured cells.     

Use of Conventional Media and Thin Agar Layer Method for Recovery of Foodborne Pathogens from Pressure-treated Poultry Products

ABSTRACT: The recovery of 5 pathogens from minced chicken (MC) and liquid whole egg (LWE) was determined with tryptone soya agar (TSA), selective medium, and thin agar layer (TAL; selective medium overlaid with TSA) to evaluate high-pressure effects at 200, 300, 400, and 500 MPa for 1 and 5 min at 20 °C. At 400 MPa for 5 min, Escherichia coli inactivation was 4 log in MC and 5.5 log in LWE, whereas no Salmonella Typhimurium, Yersinia enterocolitica , or Listeria monocytogenes were detected in either product. Staphylococcus aureus was the most pressure-resistant. In general, counts on the 3 media were not different. The TAL method allows differentiation of the inoculated pathogens from background microbiota.     

Inactivation of Listeria monocytogenes Scott A 49594 in apple juice supplemented with cinnamon

Normal (pH 3.7) and adjusted (pH 5.0) pasteurized apple juice containing cinnamon (0, 0.1, 0.2, and 0.3%) was inoculated with Listeria monocytogenes Scott A 49594 at 10(4) CFU/ml and stored at 5 and 20 degrees C for 7 days. Counts on tryptic soy agar (TSA), modified Oxford (MOX) medium, and thin agar layer (TAL) were determined at 1 h and 1, 3, and 7 days. The TAL method (MOX medium overlaid with TSA) was used for the recovery of injured cells. In apple juice, both at normal and adjusted pH, with any doses of cinnamon, no L. monocytogenes (a 4.6-log CFU/ml reduction) was detected after 1 h of storage at both temperatures, and no growth occurred at any points of storage. Therefore, cinnamon by itself (regardless of pH) had a pronounced killing effect. A further enrichment step with brain heart infusion agar showed that L monocytogenes was completely inactivated in apple juice stored at 20 degrees C, except in pH 5.0 samples with 0.1% of cinnamon. The TAL method was as effective as TSA in recovering injured cells of L. monocytogenes. Cinnamon considerably inactivates L. monocytogenes in apple juice and thus enhances the safety of this product.     

Comparison of growth kinetics for healthy and heat-injured Listeria monocytogenes in eight enrichment broths

Detection of Listeria in food products is often limited by performance of enrichment media used to support growth of Listeria to detectable levels. In this study, growth curves were generated using healthy and heat-injured Listeria monocytogenes strain F5069 in three nonselective and five selective enrichment broths. Nonselective enrichment media included the current Food and Drug Administration Bacteriological Analytical Manual Listeria enrichment broth base (BAM), Listeria repair broth (LRB), and Trypticase soy broth. Selective enrichment media included BAM with selective agents and LRB with selective agents, BCM L. monocytogenes preenrichment broth, Fraser broth, and UVM-modified Listeria enrichment broth. The Gompertz equation was used to model the growth of L. monocytogenes. Gompertz parameters were used to calculate exponential growth rate, lag-phase duration (LPD), generation time, maximum population density (MPD), and time required for repair of injured cells. Statistical differences (P < 0.05) in broth performance were noted for LPD and MPD when healthy and injured cells were inoculated into the broths. With the exception of Fraser broth, there were no significant differences in the time required for the repair of injured cells. Results indicate that the distinction between selective and nonselective broths in their ability to grow healthy Listeria and to repair sublethally injured cells is not solely an elementary issue of presence or absence of selective agents.     

Evaluation of a Double Layer Agar Plate For Direct Enumeration of Vibrio parahaemolyticus

A double layer agar plate (DLAP) was developed according to the thin agar layer (TAL) method as a 1‐step procedure for direct enumeration of injured Vibrio parahaemolyticus cells based on the formation of unique purple colonies by V. parahaemolyticus. The DLAP was prepared by overlaying an equal volume (10 mL) of a nonselective medium (tryptic soy agar supplemented with 1.5% NaCl) onto a selective medium (Bio‐Chrome Vibrio medium). The DLAP was capable of detecting V. parahaemolyticus in mixed cultures containing non‐Vibrio bacteria. Production of purple colonies by V. parahaemolyticus on DLAP was not affected by the growth of other bacteria, even when V. parahaemolyticus was only a small fraction (5%) of the entire bacterial population. Direct plating on DLAP was found as effective as the most probable number (MPN) method for recovering heat‐and cold‐injured V. parahaemolyticus cells, which could not be detected by direct plating on Bio‐Chrome Vibrio medium or thiosulfate‐citrate‐bile salts‐sucrose agar. The DLAP offers an alternative to the MPN method for detecting injured V. parahaemolyticus cells and can be used as a simple 1‐step procedure for quick screening of V. parahaemolyticus in foods.     

Modifications to methods for the enumeration and detection of injured Escherichia coli O157:H7 in foods

Reliable methods are required for the detection and enumeration of potentially injured E. coli O157 in food in the presence of outnumbering competing bacteria. Selective agents can prevent or inhibit the recovery and subsequent multiplication of injured cells and direct inoculation, either into selective enrichment broths or onto selective agar plates is still used in many methods for E. coli O157 detection and enumeration. When compared with tryptone soya agar (TSA), sorbitol MacConkey agar (SMAC) was shown to underestimate the concentration of viable E. coli O157:H7 subjected to low pH and high NaCl concentration. Using a resuscitation stage on TSA followed by membrane transfer to SMAC improved recovery to levels obtained on TSA. The membrane method was used to monitor the numbers of artificially contaminated E. coli O157:H7 during the fermentation of a meat product and demonstrated better survival when compared to counts on SMAC. Six rapid methods for the detection of E. coli O157 in food (BAX E. coli O157, Reveal 8 E. coli O157-H7 screening test, VIP EHEC, VIDAS E. coli O157 (ECO), EHEC-Tek and Tecra E. coli O157 visual immunoassay), were evaluated using beetburgers, parsley and fermented meat artificially contaminated with injured cells. Methods using direct selective enrichment, with or without an elevated incubation temperature gave false-negative results. The incorporation of a non-selective pre-enrichment medium improved the detection rates of these assays by up to ten fold.