Agar underlay method for recovery of sublethally heat-injured bacteria

A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar under laid with a selective medium. In a two-chambered petri dish, the Lutri plat e (LP), a nonselective 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 chamber of the LP and incubated. By diffusing throu gh the nonselective top agar, selective agents fi om the underlay medium im part selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salm onella typhimurium were not different (P > 0.05) from recovery rates determ ined with nonselective media. Sublethally heat-injured cells (60 degrees C for 1.5 min in buffer or 80 degrees C for 30 s on meat surfaces) grew and p roduced a typical colony morphology and color reaction when the agar underl ay procedure was used with the appropriate respective selective agars. Unli ke agar overlay methods for injury repair, the agar underlay procedure allo ws the typical selective-medium colony morphology to develop and allows col onies to be more easily picked for further characterization. Higher recover y rates of heat-injured fecal enterococci from bovine fecal samples and tot al coliforms from animal waste lagoons were obtained by the agar underlay m ethod with selective agars than by direct plating on the respective selecti ve media.