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PSEUDOMONAS AERUGINOSA EXOTOXIN ASSAY FOR STS-107 EXPERIMENT BACTER.  K.M. Hale and B.H. Pyle.  Dept. of Microbiology, Montana State University, Bozeman.

   An Exotoxin A (ETA) producing P. aeruginosa ATCC 29260 will be flown on STS-107 to study their growth, physiology and virulence.  In preparatory ground studies, ETA was analyzed by a 96-well ELISA in which standards and samples were bound to the microplate wells.  After ETA binding, a blotto solution of skim milk and bovine serum albumin in Tris-Buffer Saline-Tween was used for blocking.  A polyclonal rabbit primary antibody was added, followed by a secondary goat-HRP antibody and OPD for color development.  Each binding step was followed by rinsing with TBS and TBS-T.  Optical density was read at 490 nm on a 96-well plate reader.  For ETA quantification, standards and samples were diluted in culture medium.  The assay was optimized by adjusting standard and sample dilutions, incubation period, plating procedures, and by performing and analyzing ETA capture with the direct method of ELISA vs. an indirect method.  Following optimization, the direct assay gave reliable results with samples from ground-based experiments, with ETA concentrations in the range of 200-300 ng/ml.  Results from simulated weightlessness experiments suggest that ETA will be produced in microgravity in detectable amounts.  The flight data will assist in assessing the significance and risk of these bacteria to future space flight crew   members.  (Supported by NASA, ESA, Montana Space Grant Consortium and MSU Undergraduate Scholars Program.)