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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Meat Safety and Quality » Research » Publications at this Location » Publication #102505
Title: AGAR UNDERLAY METHOD FOR RECOVERY OF SUBLETHALLY HEAT-INJURED BACTERIA

Author
item Kang, Dong Hyun
item Siragusa, Gregory

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/9/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: Bacteria are counted by letting them grow into visible colonies on a plate of solid bacterial food known as culture media. This method is very sensitive and reliable. However, occasionally bacteria fail to grow on culture media because they have been injured, but not killed, by some antimicrobial process. That process might be, for example, application of heat, steam, chlorine, or acid. When bacteria are still living (and in some cases still able to cause disease), but are not capable of growing on culture media, they are called sublethally injured bacteria. We have developed a new and improved method of recovering sublethally injured bacteria that might be found on foods, including meat animal carcasses. This method is called the agar underlay method and uses a special culture dish known as a Lutri Plate. The agar underlay method allows sublethally injured cells to recover by first growing in a non-restrictive environment for 2 hours before underlaying the culture with a second culture medium in the Lutri Plate. This method has been shown to recover sublethally heat-injured cells of E. coli O157, as well as the disease-causing bacteria Salmonella and Listeria, from both the test tube and from beef carcass surface tissues. This method will be especially helpful to scientists trying to determine the best ways of killing pathogenic bacteria on meat animal carcasses before further processing.

Technical Abstract: A method of recovering sublethally injured bacteria was developed. The procedure (termed the agar underlay method) uses a non-selective agar underlayed with a selective medium. Using a two-chambered petri dish, the Lutri Plate(TM) (LP), a non-selective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom of the chamber of the LP and incubated. By diffusing through the non- selective top agar, selective agents from the underlay medium impart selectivity to the system. Using the agar underlay method, recovery rates of heat-injured foodborne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0.05) from recovery rates determined with non-selective media. Sublethally heat injured cells (60 deg C for 1.5 min in buffer or 80 deg C for 30 sec on meat surfaces) grew and produced a typical colony morphology and color reaction using the agar underlay procedure with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows for the typical selective medium colony morphology to develop and for colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained using the agar underlay method with selective agars than by direct plating on the respective selective media.