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Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes
Published on: August 9, 2011
Thin agar layer method for recovery of heat-injured Listeria monocytogenes
1Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506-1600, USA. kang@email.marc.usda.gov
Abstract:
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.
Insights
A novel thin agar layer (TAL) method effectively recovers heat-injured Listeria monocytogenes. This method improves bacterial enumeration compared to traditional selective media, offering a simpler alternative for food safety testing.
Area of Science:
- Microbiology
- Food Safety
- Bacteriology
Background:
- Heat-injured Listeria monocytogenes pose a significant food safety risk.
- Selective plating media like Modified Oxford medium (MOX) can inhibit the recovery of heat-stressed bacteria.
- Tryptic soy agar (TSA) allows growth but lacks selectivity.
Purpose of the Study:
- To develop and evaluate a Thin Agar Layer (TAL) method for enhanced recovery of heat-injured Listeria monocytogenes.
- To compare the efficacy of the TAL method with traditional methods (TSA, MOX, OV) for enumerating heat-stressed L. monocytogenes.
Main Methods:
- A Thin Agar Layer (TAL) method was developed by overlaying nonselective TSA onto selective MOX agar.
- Heat-injured Listeria monocytogenes in milk and peptone water were enumerated using TAL, TSA, MOX, and traditional overlay (OV) methods.
- Enumeration was performed after incubation at 37°C for 24 hours.
Main Results:
- The TAL method demonstrated no significant difference in recovery compared to TSA and the traditional overlay (OV) method.
- Both TAL and OV methods recovered significantly higher numbers of heat-injured L. monocytogenes than MOX agar.
- The TAL method is a single-step procedure, simplifying the process compared to the two-step OV method.
Conclusions:
- The Thin Agar Layer (TAL) method is effective for recovering heat-injured Listeria monocytogenes.
- TAL offers an improved and simplified alternative to existing methods for detecting stressed L. monocytogenes in food samples.
- This method enhances bacterial enumeration accuracy in food safety applications.
