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3 <br /> Endpoint Assay Processing <br /> R <br /> Strains were grown overnight(18 hours) on trypticase soy broth agar at 28 °C, and then <br /> resuspended in sterile water to a turbidity of 40%-50%T. The strains were then loaded into 96- <br /> well microtiter plates in which an undisclosed growth medium(mineral salts,vitamin mix,buffer) <br /> without a major carbon source had been previously added. The wells also contained a tetrazolium <br /> dye based redox indicator system. Bacterial growth(metabolic respiration, or oxidation of carbon <br /> sources) was monitored by tetrazolium reduction as measured at 590 nm in a microplate reader <br /> after 24 hours incubation. GASOLINE AND DIESEL FUEL WERE was added in a 25 µl volume <br /> to selected wells to serve as the major carbon source by using a multichannel pipettor to deliver a <br /> total final volume of 150 µ/well. Trypticase soy broth served as a positive growth control. <br /> Total growth was measured after 24 hours incubation at 28 °C. Data was processed and <br /> is given with background blank values subtracted A graph of the data was generated and is <br /> provided on the following page The design of the experiment is provided in conjunction with the <br /> template arrangement and positron of strains in the matrix with negative and positive control wells. <br /> Final Results: <br /> Strain 1, Strain 3 and Strain 4 grew well on diesel and gas. Strain 2 grew well on gas <br /> Disclaimer: the MiL, inc. is not a human clinical diagnostic laboratory and makes no <br /> warranty to the fitness of this data for such purposes. <br /> Thank you from the Staff on project. <br /> - Manager Bruce C.Hemming Ph.D. rations Director <br /> Juke Milke Laboratory ag g , Operations <br />