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United States Department of Agriculture

Agricultural Research Service

Title: A Rapid Two-Fold Dilution Method for Microbial Enumeration and Resuscitation of Uninjured and Sublethally Injured Bacteria

Authors
item Kang, Dong Hyun
item Siragusa, Gregory

Submitted to: Letters in Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 21, 2001
Publication Date: N/A

Interpretive Summary: It is possible for bacteria to undergo a phenomenon known as sublethal cellular injury. Injured bacteria are not dead but are unable to grow on highly restrictive or selective culture media such as those commonly used in the food microbiology lab. These organisms can then be transmitted undetected by bacterial culture tests and remain potentially harmful for humans. Many methods to resuscitate injured bacteria have been published. Most of these used solid media and are cumbersome to use. We have developed a method named the two-fold dilution method (2FD), which can both resuscitate and enumerate uninjured and sublethally injured bacterial cells, that is easy to use and does not rely on solid bacteriological media. Samples to be tested are diluted in a 96-well plastic plate in double strength selective broth. For resuscitation of heat-injured (55 deg C for 10 min) fecal bacteria, the selective broth was added to the wells after 3 h pre-resuscitation time in non- restrictive media. Results from the 2FD test were compared to standard plating methods for non-injured bacterial counts from beef carcass surface tissue samples and found not to differ. The 2FD method recovered a significantly higher number of cells that were sublethally injured by heating than did the standard plating technique. This method should be useful for laboratories wishing to rapidly enumerate potentially injured bacteria such as those having been subjected to thermal or chemical antimicrobial processes. Such processes are now common in meat processing plants and include hot water washing, steam pasteurization, and organic acid rinses.

Technical Abstract: A rapid and simple method for enumerating uninjured and sublethally injured bacterial cells, the two-fold dilution method (2FD), was developed and evaluated. Following two-fold serial dilution of samples in a 96 well microtiter plate, double strength selective broth on non-selective broth was added to each well. For resuscitation of heat-injured (55 deg C for 10 min) coliforms, the selective broth was added to the wells after 3 h pre-resuscitation time in buffered peptone water. The results of the 2FD were compared to plating methods for total and coliform plate counts from mixed cultures and beef carcass surface tissue samples. The 2FD method results were not significantly different (P > 0.05) from those obtained using Petrifilm and standard plating. Correlation of the scatterplot of spread plating and 2FD indicated a high level of agreement between these two methods (R**2 = 0.98 for total counts and R**2 = 0.96 for coliforms from mixed cultures; R**2 = 0.98 for total cell counts and R**2 = 0.94 for coliforms from feces inoculated beef carcasses). The two-fold dilution method recovered significantly higher numbers of heat- injured coliforms compared to conventional plating methods (P < 0.05).

Last Modified: 4/17/2014
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