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OPTIMIZATION OF PSEUDOMONAS AERUGINOSA INOCULUM FOR STS-107 EXPERIMENT BACTER.  A.C. Perez-Osorio, S.C. Broadaway, and B.H. Pyle.  Microbiology Department, Montana State University, Bozeman.

   The growth, physiology and virulence of an Exotoxin A (ETA) producing isolate of P. aeruginosa will be studied in this flight experiment.  A suspension of 5x10⁸ cells/ml is required to ensure growth of defined medium cultures in ESA Phorbol cassettes with Type I containers.

   An initial culture was grown in Simple Defined Medium for18-24 hours. When the culture had reached between 60-100 Klett Units the Optical Density (OD) was also determined.  After washing twice in PBS-glucose, the suspension was diluted to greater than 5x10⁸ cells/ml based on the OD. The sample was then adjusted to  5x10⁸ cells/ml according to SYBR Green staining with microscopic enumeration. The resulting suspensions were either enumerated immediately or stored at 5⁰C for 7 days and analyzed to enumerate the surviving population and viability of the inoculum.  Based on the results, SDM cultures for the space flight experiment will be grown to 0.5-0.6 OD prior to washing and inoculum preparation, rather than from the amount of time incubated. The inoculum survived the  7 day storage time, and subsequent inoculation of ETA medium produced growth as required.

   Using an inoculum prepared as described, we anticipate reliable inoculation and growth of ETA medium cultures in the spaceflight hardware.  The flight experiment will improve our understanding of the significance of P. aeruginosa to future spaceflight crews.

(Supported by NASA, ESA, MSU Undergraduate Scholars Program, and Montana Space Grant Consortium)