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Applied and Environmental Microbiology, December 1999, p. 5334-5337, Vol. 65, No. 12
Roman L. Hruska U.S. Meat Animal Research
Center, Agricultural Research Service, United States Department of
Agriculture, Clay Center, Nebraska 68933-0166
Received 7 June 1999/Accepted 7 September 1999
A method of recovering sublethally heat-injured bacteria was
developed. The procedure (termed the agar underlay method) uses a
nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (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 through the nonselective top
agar, selective agents from the underlay medium impart selectivity to
the system. By the agar underlay method, recovery rates of the
heat-injured food-borne pathogens Escherichia coli O157:H7
and Salmonella typhimurium were not different
(P > 0.05) from recovery rates determined with
nonselective media. Sublethally heat-injured cells (60°C for 1.5 min
in buffer or 80°C for 30 s on meat surfaces) grew and produced a
typical colony morphology and color reaction when the agar underlay
procedure was used with the appropriate respective selective agars.
Unlike agar overlay methods for injury repair, the agar underlay
procedure allows the typical selective-medium colony morphology to
develop and allows 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 by the agar underlay method with selective
agars than by direct plating on the respective selective media.
0099-2240/99/$04.00+0
Agar Underlay Method for Recovery of Sublethally
Heat-Injured Bacteria
*
Corresponding author. Mailing address: Hruska U.S. Meat
Animal Research Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 166, Spur 18D, Clay Center, NE 68933-0166. Phone: (402) 762-4227. Fax: (402) 762-4149. E-mail:
siragusa{at}emailmarc.usda.gov.
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