Citation:

Donegan, K., C. Matyac, R. Seidler, AND A. Porteous. EVALUATION OF METHODS FOR SAMPLING, RECOVERY, AND ENUMERATION OF BACTERIA APPLIED TO THE PHYLLOPANE. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-91/031 (NTIS PB91182303).

Description:

Determining the fate and survival of genetically engineered microorganisms released into the environment requires the development and application of accurate and practical methods of detection and enumeration. everal experiments were performed to examine quantitative recovery methods that are commonly used or that have potential applications. n these experiments, Erwinia herbicola and Enterobacter cloacae were applied in greenhouses to Blue Lake bush beans (Phaseolus vulsaris) and Cayuse oats (Avena sativa). ampling indicated that the variance in bacterial counts among leaves increased over time and that this increase caused an overestimation of the mean population size by bulk leaf samples relative to single leaf samples. n increase in the number of leaves in a bulk sample, above a minimum number, did not significantly reduce the variance between samples. xperiments evaluating recovery methods demonstrated that recovery of bacteria from leaves was significantly better with stomacher blending, than with blending, sonication, or washing and that the recovery efficiency was constant over a range of sample inoculum densities. Delayed processing of leaf samples, by storage in a freezer, did not significantly lower survival and recovery of microorganisms when storage was short term and leaves were not stored in buffer. he drop plate technique for enumeration of bacteria did not significantly differ from the spread plate method. esults of these sampling, recovery, and enumerations experiments indicate a need for increased development and standardization of methods used by researchers as there a significant differences among, and also important limitations to, some of the methods used.

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