research article evaluation of the thin agar layer oxyrase
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Central Journal of Human Nutrition & Food Science
Cite this article: Wu VCH, Fung DYC (2014) Evaluation of the Thin Agar Layer OxyraseÂź Method for Recovery of Heat-Injured Foodborne Pathogens in Liquid Media and Food Systems. J Hum Nutr Food Sci 2(2): 1027.
*Corresponding authorVivian CH Wu, School of Food and Agriculture, Pathogenic Microbiology Laboratory, The University of Maine, 5395 Hitchner Hall, Orono, ME 04469-5735, USA, Tel: 1-207-5813101; Fax:1-207-5811636; E-mail:
Submitted: 10 October 2013
Accepted: 10 December 2013
Published: 03 January 2014
ISSN: 2333-6706
Copyright© 2014 Wu et al.
OPEN ACCESS
Research Article
Evaluation of the Thin Agar Layer OxyraseÂź Method for Recovery of Heat-Injured Foodborne Pathogens in Liquid Media and Food SystemsVivian CH Wu1* and Daniel YC Fung2
1School of Food and Agriculture, Pathogenic Microbiology Laboratory, The University of Maine, USA2Department of Animal Sciences and Industry, Kansas State University, USA
INTRODUCTIONRecovery of injured microorganisms from processed food
and environmental samples has been a major concern of applied bacteriologists [1]. Injured cells are cells that survive a stress but lose some of their distinctive qualities [2]. As opposed to a dead cell, an injured cell can repair the cellular damage (resuscitation) when supplementing the restricted medium with specific nutrients and then regain its ability to form a colony in the presence of the selective agent; however, the dead cell cannot form a colony under any conditions [3]. Pathogens and spoilage organisms in foods can become injured within food products after processing and handling procedures. Injury to pathogens eliminates their ability to cause disease, but once the cells are repaired pathogenicity is totally restored [4]. Therefore, determining the presence of impaired microorganisms is important in all aspects of food processing and preservation [2].
The utility of a sterile suspension of bacterial membrane fragments and their associated enzymes as reagents for elimination of dissolved oxygen was first reported by Adler and Crow [5]. After the addition of membrane fractions, the redox potential in a medium is reduced to -200 to -300 mV becoming
completely anaerobic within about a minute. The partially purified membrane fragments from Escherichia coli are now commercially available as Oxyrase (Oxyrase, Inc., Mansfield, OH). A unit of OxyraseÂź per milliliter reduces dissolved oxygen at the rate of 1% per second at 37 oC, pH 8.4 in 40 mM phosphate buffer and 50 mM sodium lactate in an air saturated solution. The activity of OxyraseÂź increases with increased temperature up to 55oC and the effective range of pH is from 6 to 9. Oxygen reducing membrane or OxyraseÂź has been used to remove dissolved oxygen from media for culturing anaerobes and to stimulate growth of pathogenic facultative anaerobes. It also improves growth of heat-, cold-, chemical-, and radiation-injured microorganisms [6-14]. Yu and Fung [15] reported that heat-injured and healthy cells of L. monocytogenes were significantly recovered in Fraser Broth supplemented with 0.01 units/ml of OxyraseÂź compared with control broth without the supplement. Oxygen is potentially toxic to injured cells [16]. Dubos [17] and Nelson [18] indicated that the low redox potential was responsible for allowing growth of heat-injured facultative microorganisms. Knabel and others [19] reported that use of the strictly anaerobic Hungate technique significantly increased recovery of heat-injured L. monocytogenes compared with aerobically incubated controls.
Abstract
A method called Thin Agar Layer OxyraseÂź Method, which is Tryptic Soy Agar with 1:30 dilution of âOxyraseÂź for Agarâ overlaid onto a pre-poured and solidified pathogen-specific selective medium was developed. This method improved recovery of heat-injured Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Yersinia enterocolitica individually in liquid medium. We evaluated the TALO method for recovery of the above four heat-injured (55oC, 10 min) pathogens in a mixture environment, as well as in food systems (ground beef and milk). The TALO method recovered greater numbers of pathogens (P>0.05) than selective media for four pathogens from all conditions, thus providing more sensitive detection levels for practical applications.
KeywordsâąOxyraseÂźâąHeat-Injured Pathogens
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OxyraseÂź could be considered as a supplement to the resuscitation Thin Agar Layer system (non-selective agar overlaid on selective agar) [20-22] to increase the capability of recovery for injured pathogens. We have developed the Thin Agar Layer Oxyrase Method (Tryptic Soy Agar with 1:30 dilution of âOxyraseÂź for Agarâ overlaid onto a prepoured and solidified pathogen-specific, selective medium in a petri dish). This method improved the capability for recovery of heat-injured Yersinia enterocolitica, Salmonella Typhimurium, L. monocytogenes, and E. coli O157:H7 individually in pure culture liquid medium. The objective of this study was to evaluate and apply the Thin Agar Layer Oxyrase Method (TALO) for the detection of injured foodborne pathogens from a mixture of Y. enterocolitica, Salmonella Typhimurium, L. monocytogenes, and E. coli O157:H7 in liquid medium, as well as solid food using ground beef (22% fat) and liquid food using milk samples (skim milk).
MATERIALS AND METHODSCulture preparation
Four foodborne pathogens were used: E. coli O157:H7 (American Type Culture Collection, ATCC 35150), L. monocytogenes (ATCC 49594), S. Typhimurium (United States Department of Agriculture, USDA), and Y. enterocolitica virulent serotypes O:8 (National Animal Disease Center, NADC). All cultures were checked for purity and authenticity by commercial diagnostic kits (API 20E, latex agglutination test and Crystal ID systems). Each culture was grown in 9.0 ml Tryptic Soy Broth and incubated at 37oC for 24 hrs. After incubation, 1 ml of each culture broth were transferred to sterilized 500-ml polycarbonate centrifuge bottles (Nalgene, Rochester, NY) containing 100 ml of BHI broth, and the bottles were incubated at 37oC for 24 hrs. Cells were harvested by centrifugation (Model JA-14, International Equipment Co., Needham Heights, MA) at 15,300 x g for 10 min at 4oC. After centrifugation, the pellet was resuspended in 0.1% peptone water. The resuspended cells were again centrifuged and resuspended and the resulted culture suspensions were used for experiments [23,24]. For the study of mixed cultures in liquid medium, cells were adjusted to approximately 7 log CFU/ml individually and a pathogen cocktail was made by combining four individual diluted cultures (250 ”l of each culture) into a sterile tube. For the study of food samples, the numbers of cells were adjusted to approximately 8 log CFU/ml by diluting with 0.1 % sterile peptone water.
Media and recovery methods
To enumerate both injured and uninjured pathogens, Tryptic Soy Agar (TSA, Difco, Detroit, MI) was used as a basic nonselective medium. Selective media were MacConkey Sorbitol Agar (MSA, Difco) for E. coli O157:H7, Modified Oxford (MOX, Difco) for L. monocytogenes, Xylose Lysine Desoxychlolate (XLD, Difco) for S. Typhimurium, and Cefsulodin-Irgasan-Novobiocin (CIN, Oxoid, England) for Y. enterocolitica. Single TAL plates were made by overlaying a total of 14 ml of nonselective medium (TSA) onto a prepoured and solidified pathogen-specific, selective medium in a petri dish (TSA/CIN, TSA/XLD, TSA/MOX, and TSA/MSA; detail was described in 20-22).
Thin Agar Layer OxyraseÂź (TALO) Method was developed by overlaying a total of 14 ml of TSA with 1:30 dilution of
âOxyraseÂź for Agarâ onto a prepoured and solidified pathogen-specific, selective medium in a petri dish (100x15mm). TSA was autoclaved before addition of Oxyrase. After cooling, TSA to 45oC to 48oC, 1:30 dilution of âOxyraseÂź for Agarâ was added and mixed well as TSAO (TSA with OxyraseÂź). After solidification of the sterilized selective agar in a petri dish, 7 ml of melted TSAO (48oC) was overlaid. One minute after solidification of the first 7 ml layer of TSAO, a second 7 ml layer of TSAO was overlaid. A four-compartment petri dish (100x15mm, Fisher Scientific, Pittsburgh, PA) was also used for Thin Agar Layer Oxyrase (TALO) method. Nine ml of four pathogen-specific, selective media (CIN, XLD, MOX, and MSA) were poured into individual compartments of the petri dish. After solidification of the sterilized selective agar, 11 ml of melted TSAO (48oC) were laid over all four selective agars to form a continuous thin agar layer. One minute after solidification of the first 11-ml layer of TSAO, a second 11-ml layer of TSAO was overlaid, for a total of 22 ml of TSAO [25]. Comparative analysis of enumeration of noninjured and injured pathogens was made among pathogen selective agar, the single TAL plates, the single TALO plates and the four-compartment TALO (4-TALO) plates for both liquid medium and food system studies. Nonselective medium (TSA) provided the total counts of the mixed pathogens.
Recovery of heat-injured foodborne pathogens from a mixture in 0.1 % Peptone water
One ml of the pathogen cocktail (diluted to about 7.0 log CFU/ ml) was added to a screw-cap test tube containing 9 ml of 0.1% peptone water. The tubes and peptone water had been preheated and maintained at 55oC. The cap of the tube was tightly screwed, and the portion of the tube containing liquid was completely immersed in a 55oC water bath shaker and heated for 10 min [24,26,27]. A thermometer was used to measure the temperature of water in an uninoculated tube to ensure accuracy of heating at 55oC. After heating, the tubes with the pathogen suspension were removed from the water bath and cooled immediately in slush ice to room temperature (25oC, 10 min). Unheated samples were used as the control. After serial 10- fold dilutions using 0.1% sterile peptone water, individual pathogen suspensions were spiral plated onto duplicate TSA, pathogen selective agar, TAL, TALO and 4-TALO media with a Spiral Plater (Spiral Biotech. Inc.,Bethesda MD). Plates were incubated at 37oC for 24 hrs.
Recovery of Heat-injured Pathogens from Ground Beef
A pathogen cocktail was made by combining four individual cultures in a sterile tube (2.5 ml of each culture). After vortexing the mixtures well, 90 g of fresh ground beef purchased from a local grocery store (22% fat) were inoculated with 10 ml of a pathogen cocktail (approximately 8 log CFU/ml) to achieve initial inoculum level of approximately 7 log CFU/g and thoroughly mixed for 2 minutes by manual massaging. The beef sample was set at room temperature for 1 hr to assist absorption of inocula. Three 25-g portions of sample were added to a sterile filter stomacher bag (Spiral Biotech, Inc., Bethesda, MD) and immersed in a shaking water bath. Each 25-g sample was spread evenly in the bag to facilitate uniform heat transfer during heat treatment. Parameters for injury were set at 55°C for 10 minutes.
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A thermometer was used to measure the temperature of meat (25 g) in an uninoculated stomacher bag to ensure accuracy of heating at 55oC. After heating, the samples were cooled in an ice bath until the beef has reached room temperature (ca 25oC, ca 10 min). Unheated samples served as controls. Two hundred and twenty five ml of 0.1% peptone was added to each filter stomacher bag and the samples were stomached for 2 minutes with the Stomacher (Seward Medical London, UK) [28]. After serial 10-fold dilutions using 0.1% sterile peptone water, suspensions were spiral plated onto duplicate nonselective (TSA) agar, pathogen selective agar, TAL, TALO and 4-TALO media with a Spiral Plater (Spiral Biotech, Inc., Bethesda, MD). Plates were incubated at 37oC for 24 hrs.
Recovery of Heat-injured Pathogens from Milk
Four individual cultures (250 ”l of each culture) were combined into a sterile tube and vortexed. One ml of pathogen cocktail approximately 8 log CFU/ml was spiked into 99 ml of skim milk (approximately 6 log CFU/ml). The milk sample was set at room temperature for 1 hr to facilitate absorption of inocula. Three 10-ml portions of sample were heat-treated and monitored at 55oC for 10 min [23] as previous described. After cooling in slush ice, milk samples were diluted serially with 0.1% peptone water and spiral plated onto duplicate nonselective (TSA) agar, pathogen selective agar, TAL, TALO and 4-TALO media with a Spiral Plater (Spiral Biotech, Inc., Bethesda, MD). Plates were incubated at 37oC for 24 hrs. Unheated samples served as controls were spiral plated onto the same medium method as heated samples after serial dilutions.
Data analysis
The recovery capacity was compared among pathogen selective agar, TAL, TALO, and 4-TALO except TSA in liquid medium and food samples. TSA could not differentiate target pathogen from mixed flora and only provided total counts of the mixtures in the mixed culture study. The experiments were repeated three times. The percentage of increased recovery was calculated by the formula: Percent increased recovery = [(counts on recovery medium â counts on selective medium)/counts on selective medium] x 100 using actual bacterial numbers counted from the media [29-31]. Bacterial numbers were also converted to log10CFU/ml for statistical analysis. The experiment design was a Randomized Complete Block (RCB). Analysis of variance (ANOVA) was performed on cell counts using the SAS General Linear Models (GLM) procedure with SAS software version 6.12 (SAS Institute, Cary, NC). Means of three replicates were plotted in graphs, and significant differences determined at the 95% confidence limit. Least Square Difference (LSD) test was employed to quantify significant differences among means of bacterial numbers that were transformed to log10CFU/ml prior to analysis. Fisherâs confidence interval was calculated as:
( . .i jY Yâ ) ± LSD, where .iY and .jY are two means for pairwise comparison [32].
RESULTS AND DISCUSSION
Mixed cultures in liquid medium
According to our previous studies, the Thin Agar Layer
OxyraseÂź Method was an improvement over the existing Thin Agar Layer Method when evaluated for individual cultures in a liquid medium (0.1% peptone water). The TALO method recovered greater numbers of pathogens than selective media as well as the TAL method did. In addition, the TALO method had better recovery than the existing TAL method when compared with pathogen-specific selective media. In this study, E. coli O157:H7, L. monocytogenes, S. Typhimurium, and Y. enterocolitica were studied as a mixture and recovery capacity was compared among pathogen selective agar; TAL, TALO, and 4-TALO for detecting target pathogen form the mixed cultures.
This mixed culture study also showed that both TALO and TAL methods recovered injured cells, provided nutrients for injured organisms to grow on the top layer (nonselective agar, TSAO in the TALO method, TSA in the TAL method). Then the selective agents from the bottom layer (selective agar) diffused into TSA to form typical reaction colonies with the target microorganisms. Neither the TSAO nor the TSA layer influenced the typical colors of colonies produced by target microorganisms in the mixtures. Both TALO and TAL methods recovered greater numbers of pathogens than selective media. Moreover, the TALO method showed increased recovery than the existing TAL method when compared with pathogen-specific selective media. The TALO method was also applied to a 4-compartment petri dish (4-TALO). It showed greater recovery capability than pathogen-specific selective media and higher percent increase of recovery than the TAL method. The 4-TALO system was able to recover and distinguish, on the appropriate compartment, each individual pathogen from the 4-pathogen mixture. Moreover, it would allow simultaneous selective recovery of all four pathogens studied, improving the overall efficiency of the single-TALO and TAL systems.
For heat-injured (55oC/10 min) E. coli O157:H7 from a mixture (E. coli O157:H7, L. monocytogenes, S. Typhimurium, and Y. enterocolitica) TALO (TSAO/MSA), 4-TALO (4-TSAO/MSA) and TAL (TSA/MSA) showed similar increases in recovery. The recovery capacity of MSA was increased by TALO by 365%; by 4-TALO with 346%; and by TAL with 332%. When recovered cells were analyzed in the form of logarithm (Figure 1a), TALO, 4TALO, and TAL recovered more (approximately 1 log) (P<0.05) heat-injured E. coli O157:H7 than MSA. The 95% confidence interval was from â0.09 to 0.15 log for the difference between TALO and TAL (TALO-TAL). The TALO method did not improve the existing TAL method greatly for heat-injured E. coli O157:H7 from the mixed cultures, indicating either method could be used as the recovery method and replace the commercial selective medium, MSA. However, considering both injured and non-injured cells, OxyraseÂź in TALO would facilitate the enumeration of organisms in terms of actual cell numbers. TALO, 4-TALO and TAL distinguished E. coli O157:H7 as clear colorless colonies.
Heat-injured L. monocytogenes was recovered well by TALO (TSAO/MOX) with the highest percent increase of recovery, 821% when compared with the selective medium, MOX. The second highest percent increase of recovery was 4-TALO (4-TSAO/MOX) with 786%, followed by TAL (TSA/MOX) with a 531% increase. Figure 2a shows that TALO, 4-TALO, and TAL had greater recovery of heat-injured L. monocytogenes, (approximately 1 log) than MOX (P<0.05). The 95% confidence interval for the
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Figure 1 Comparison of TSA, selective media, TAL, TALO and 4-TALO for recovery of heat-injured (55 oC/10 min) E. coli O157:H7 from the mixed culture in a) 0.1% peptone water, b) ground beef, and c) in milk.
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difference between the TALO and TAL methods was â0.18 to 0.47 log. The TAL and TALO methods differentiated L. monocytogenes as black colonies.
Compared with XLD, greater recovery of heat-injured S. Typhimurium was observed for 4-TALO (4-TSAO/XLD, 2842%), TALO (TSAO/XLD, 2636%) and then TAL (TSA/XLD, 1767%). The TALO method (TALO or 4-TALO) gave approximately 1.6 times higher recovery than the TAL method. In the analysis of log numbers (Figure 3a), all 4-TALO, TALO and TAL showed greater recovery (approximately 1-1.5 log) of heat-injured S. Typhimurium than XLD (P<0.05). The 95% confidence interval for the difference between TALO and TAL was â0.45 to 0.76 log. TALO, 4-TALO and TAL distinguished S. Typhimurium as black colonies.
The percent increase of recovery for heat-injured Y. enterocolitica as compared with CIN was 12218%, 5285% and 2733% for 4-TALO, TALO and TAL, respectively. The 4-TALO (4-TSAO/CIN) and TALO (TSAO/CIN) showed approximately 2 to 4.5 times greater percent increase of recovery than TAL. This indicated an improvement over the TAL method by the TALO method for heat-injured Y. enterocolitica. Comparison in log numbers showed a significantly greater recovery of Y. enterocolitica for 4-TALO, TALO and TAL (approximately 1-1.5 log) over CIN (P<0.05) (Figure 4a). The 95% confidence interval was â0.52 to 1.14 log for the difference between TALO and TAL (TALO-TAL). TALO, 4-TALO, and TAL differentiated Y. enterocolitica as pink colonies.
Considering enumeration of both non-injured and injured pathogens, the TALO method showed increased recovery over the existing TAL method, thus providing more sensitive detection levels than the TAL method.
Recovery of heat-injured pathogens from ground beef
All four injured pathogens were recovered well by the TALO and TAL methods, indicating greater recovery of sublethally injured organisms than selective media. The 4-TAL system was able to recover and distinguish each individual pathogen on the appropriate compartment from the 4-pathogen mixture in ground beef.
Heat-injured (55oC/10 min) E. coli O157:H7 was recovered with highest percent increase of recovery by TALO (TSAO/MSA, 290%), followed by 4-TALO (4-TSAO/MSA, 213%), and then TAL (TSA/MSA, 178%) when compared with MSA. The TALO method showed a slight improvement over the TAL method for recovery of heat-injured E. coli O157:H7 cells in ground beef samples. The analysis of log numbers in (Figure 1b) indicated TALO, 4TALO, and TAL recovered better than MSA (P<0.05). The 95% confidence interval for the difference between TALO and TAL was â0.06 to 0.31 log. Compared with the selective medium MOX, heat-injured L. monocytogenes was recovered well by 4-TALO (TSAO/MOX) with the highest percent increase of recovery, 5424%. The second highest percent increase of recovery was TALO (TSAO/MOX) with a 4599% increase, followed by TAL (TSA/MOX) with a 4434% increase. All these methods, 4-TALO, TALO and TAL, recovered more heat-injured L. monocytogenes (approximately 1.3-1.8 log) from the pathogen mixture in ground beef than MOX (P<0.05) (Figure 2b). The 95% confidence interval
for the difference between the TALO method and the TAL method was â0.30 to 1.71 log.
Greater percent increase of recovery of heat-injured S. Typhimurium as compared with XLD was observed for 4-TALO (4-TSAO/XLD) with 58233%. TALO (TSAO/XLD) had a 3993% increase and TAL (TSA/XLD) had a 3063% increase. In the analysis of log numbers (Figure 3b), 4-TALO, TALO and TAL showed greater recovery (approximately 1-1.6 log) of heat-injured S. Typhimurium than XLD (P<0.05) from the mixed cultures in the ground beef samples. The 95% confidence interval for the difference between the TALO method and the TAL method was â0.22 to 1.24 log.
Both 4-TALO (4-TSAO/CIN) and TALO (TSAO/CIN) showed greater recovery than TAL for heat-injured Y. enterocolitica when compared with the selective medium, CIN. The percent increase of recovery as compared with CIN was 1419%, 1207% and 447% for 4-TALO, TALO and TAL, respectively. This indicated that the existing TAL method was greatly improved by the addition of OxyraseÂź (TALO method). Figure 4b indicated that greater (P<0.05) recovery of Y. enterocolitica was observed for 4-TALO, TALO and TAL over CIN (P<0.05) and both TALO methods were also statistically better than TAL.
Recovery of heat-injured pathogens from milk
The TALO method showed 1.1 to 5.7 times improvement over the TAL method. Moreover, when the TALO method was applied on the four-compartment petri dishes, the system was able to recover and distinguish each individual pathogen on the appropriate compartment from the 4-pathogen mixture in milk samples.
The recovery capacity of MSA was increased by TALO with 264%, followed by 4-TALO with 238% and then TAL with 224% for recovery of heat-injured (55oC/10 min) E. coli O157:H7 from a mixture in milk samples. In the analysis of logarithm-recovered cells (Figure 1c), TALO and 4-TALO recovered injured E. coli O157:H7 better than MSA (P<0.05), while TAL was not different from MSA (P>0.05). This supports the assertion that an improvement over TAL has been made by the TALO method.
Heat-injured L. monocytogenes was recovered well by TALO (TSAO/MOX) with the highest percent increase (587%) of recovery over MOX. The second highest was TAL (TSA/MOX) with a 427% increase, followed by 4-TAL (4-TSAO/MOX) with a 346% increase. Figure 2c shows that TALO, 4-TALO, and TAL had greater recovery of heat-injured L. monocytogenes than MOX (P<0.05).
Compared with XLD, greater percent increase of recovery of heat-injured S. Typhimurium was observed for 4-TALO (4-TSAO/XLD, 813%), TALO (TSAO/XLD, 557%) and then TAL (TSA/XLD, 516%) (Figure 3c). In the analysis of log numbers all 4-TALO, TALO and TAL showed greater recovery (approximately 1 log) of heat-injured S. Typhimurium than XLD (P<0.05). The 95% confidence interval for the difference between TALO and TAL was â0.25 to 0.62 log.
Greater percent increase of recovery for heat-injured Y. enterocolitica as compared with CIN was obtained from TALO
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Figure 2 Comparison of TSA, selective media, TAL, TALO and 4-TALO for recovery of heat-injured (55oC/10 min) L. monocytogenes from the mixed culture in a) 0.1% peptone water, b) ground beef, and c) in milk.
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Figure 3 Comparison of TSA, selective media, TAL, TALO and 4-TALO for recovery of heat-injured (55 oC/10 min) S. Typhimurium from the mixed culture in a) 0.1% peptone water, b) ground beef, and c) in milk.
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Figure 4 Comparison of TSA, selective media, TAL, TALO and 4-TALO for recovery of heat-injured (55 oC/10 min) Y. enterocolitica from the mixed culture in a) 0.1% peptone water, b) ground beef, and c) in milk.
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(TSAO/CIN) and 4-TALO (4-TSAO/CIN) with 3617% and 2428%, respectively (Figure 4c). The TALO method recovered more than the TAL (TSA/CIN, 639%) method. Both TALO and 4-TALO showed greater recovery of Y. enterocolitica than CIN (P<0.05) while there was no significant difference between TAL and CIN (P>0.05). The 95% confidence interval was â0.06 to 1.23 for the difference between TALO and TAL.
In this study, a Thin Agar Layer OxyraseÂź Method was used to recover heat-injured foodborne pathogens. This method uses OxyraseÂź, which reduces oxygen in the environment during the resuscitation of injured-cells. Oxygen may become toxic for facultative anaerobic pathogens when cells are injured. When detecting pathogens in food samples, OxyraseÂź worked with the nonselective medium (TSA) to enumerate injured and non-injured anaerobes as well as facultative anaerobes, then the selective agents from the bottom layer (selective medium) diffused into TSAO to inhibit background flora but allowed the target recovered cells to produce colonies with typical reaction color. The whole system combined both sensitive enumeration and good differentiation for detecting pathogens in the food samples. Moreover, applying the TALO method in a four-compartment petri dish is at least four times more efficient than the single plate system because it enumerated four pathogens at the same time [25].
CONCLUSIONSThe Thin Agar Layer OxyraseÂź Method was developed as a
modification of the TAL method and has shown better percent increase of recovery than the existing TAL method, thus providing more sensitive detection than the TAL method for practical applications in mixed cultures and food samples as shown in the present study. The procedure of the TALO method is also less cumbersome and complicated compared to the traditional two-step overlay method, which requires a long wait between each manipulation. The improvement for the TALO methods has been demonstrated to be more effective for the detection of both non-injured and injured pathogens and is more rapid and reliable than the traditional methods. Moreover, applying the TALO method with a four-compartment plate allows simultaneous selective recovery of all four pathogens studied and is more efficient than the single plate system, considering the efficiency of operation and savings in material, space and labor.
ACKNOWLEDGEMENTSThis research was supported by the Maine Agricultural
and Forest Experiment Station at the University of Maine with external publication number 3360 and Kansas Agricultural Experiment Station. This work is based upon research supported in part by Hatch Grant number ME08562-10 from the USDA National Institute of Food and Agriculture.
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Wu VCH, Fung DYC (2014) Evaluation of the Thin Agar Layer OxyraseÂź Method for Recovery of Heat-Injured Foodborne Pathogens in Liquid Media and Food Systems. J Hum Nutr Food Sci 2(2): 1027.
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