Capability of Selective Media to Detect Heat-Injured Shigella flexneri
1U.S. Department of Agriculture, ARS, Eastern Regional Research Center, 600 East Mermaid Lane, Philadelphia, PA 19118.
Abstract:
Heat-injured cells of Shigella flexneri 5348 were detected by using a two-media plating system: tryptic soy broth containing 2% agar plus 1% sodium pyruvate (TSBA + P) on which both injured and non-injured cells produce colonies and semisynthetic agar medium plus 0.15% bile salts #3 (SSBA) on which only non-injured cells grew. Subtracting the count on SSBA from that on TSBA + P gave an estimation of injury. Heating washed cells of S. flexneri in phosphate buffer gave about 3 log units of injury after 40 min at 52°C. A number of broths and agars specific for shigellae and gram-negative bacteria (2% agar was added to selective broths) were tested for their ability to supported colony formation of heat-injured S. flexneri . Only one medium, shigella broth containing novobiocin, gave recovery of heat-injured cells similar to that on TSBA + P. Lauryl tryptose broth, Levine EMB agar, and EMB agar were the least inhibitory to repair of injured cells (as compared to SSBA). Media that were more inhibitory to repair of heat-injured cells (but not as inhibitory as was SSBA) included tergitol 7 agar, desoxycholate lactose agar, brilliant green 2% bile broth, GN broth, Mac-Conkey agar, and Endo agar. Desoxycholate agar, violet red bile agar, SS agar, and desoxycholate citrate agar were more inhibitory than SSBA to repair. The addition of 1% sodium pyruvate to MacConkey agar, desoxycholate lactose agar, lauryl tryptose broth, tergitol 7 agar, EMB agar, and SSBA resulted in increased recoveries of heat-injured S. flexneri but did not lead to complete recovery (i.e., the counts on the pyruvate containing selective media did not give counts equal to TSBA + P). These results indicate that most media used for isolation of shigellae do not permit recovery of heat-injured cells, and special techniques must be incorporated into the isolation procedures to recover injured cells.
Insights
Most Shigella isolation media inhibit heat-injured cells. Adding sodium pyruvate to some media improved recovery, but specialized methods are needed to detect heat-stressed Shigella flexneri.
Area of Science:
- Microbiology
- Food Safety
- Bacteriology
Background:
- Heat injury significantly impacts bacterial viability and detection.
- Standard microbiological media often fail to support the recovery of stressed or injured bacterial cells.
- Accurate detection of heat-injured Shigella flexneri is crucial for food safety and public health.
Purpose of the Study:
- To evaluate the recovery of heat-injured Shigella flexneri on various selective and non-selective media.
- To determine the inhibitory effects of common isolation media on heat-stressed bacteria.
- To assess the efficacy of sodium pyruvate supplementation in enhancing the recovery of injured Shigella.
Main Methods:
- A two-media plating system was employed: Tryptic Soy Broth with 2% agar and 1% sodium pyruvate (TSBA + P) for total viable counts, and a semisynthetic agar medium with 0.15% bile salts #3 (SSBA) for non-injured cells.
- Heat injury was induced in Shigella flexneri cells by heating in phosphate buffer at 52°C for 40 minutes.
- Various selective media for Gram-negative bacteria and Shigella were tested for their ability to support the growth of heat-injured cells, with and without sodium pyruvate supplementation.
Main Results:
- Heating Shigella flexneri cells resulted in approximately 3 log units of injury.
- Only Shigella broth with novobiocin effectively recovered heat-injured cells, similar to TSBA + P.
- Most tested media, including MacConkey agar and Endo agar, were inhibitory to the repair of heat-injured cells, with SSBA being highly inhibitory.
- Supplementation with 1% sodium pyruvate increased the recovery of heat-injured Shigella flexneri on several media but did not achieve complete recovery.
Conclusions:
- Standard media used for Shigella isolation are generally unsuitable for recovering heat-injured cells.
- The addition of sodium pyruvate can partially mitigate the inhibitory effects of some media on injured bacteria.
- Specialized techniques or media modifications are essential for the accurate detection and enumeration of heat-stressed Shigella flexneri in food and environmental samples.
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