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Cyto <br /> Culture <br /> ENVIRONMENTAL <br /> BIOTECHNOLOGY <br /> now <br /> CytoCulture International,Inc. <br /> 249 Tewksbury Avenue <br /> Pt.Richmond,CA 94801 USA <br /> PSC Industrial Outsourcing, LP Reporting Date: July 7,2009 <br /> 210 West Sand Bank Road CytoCulture lab login: 09-150 <br /> Columbia, IL 62236 Project number: 62403335-024522 <br /> Tel: (618) 281-1543 Fax: <br /> Project Manager: Paul Anderson <br /> E-mail: panderson@pscnow.com <br /> Samples: One soil sample on ice was received 06/25/09.It was stored at 4°C and assayed on the <br /> same day. Please see attached chain of custody. <br /> AEROBIC <br /> Hydrocarbon-Degrading and Heterotrophic Bacteria Enumeration Assays <br /> Analysis Request: Enumeration of aerobic petroleum hydrocarbon-degrading bacteria (broad range <br /> petroleum derived from gasoline and diesel) and aerobic total heterotrophic bacteria by method <br /> 9215A (HPC)/ Standard Methods 9215B modified. <br /> Carbon Source for Aerobic Hydrocarbon-Degrading Bacteria: Pasteurized Chevron No. 2 <br /> gasoline and diesel were dissolved into agar plates as the sole carbon and energy source for the <br /> growth of aerobic hydrocarbon-degrading bacteria. <br /> Carbon Source for Aerobic Total Heterotrophic Bacteria: Growth medium was prepared with <br /> standard methods total plate count agar(Difco) containing a wide range of carbon sources derived <br /> from yeast extract,tryptone, pancreatic digest of casein and glucose. <br /> Protocol for Hydrocarbon-Degrading Bacteria: Sterile agar plates (100 x 15 mm)were prepared <br /> with minimal salts medium at pH 6.8 with agar and hydrocarbons, without any other carbon sources <br /> or nutrients added. Triplicate plates were inoculated with 1.0 ml of a 0.2%sodium pyrophosphate <br /> extract of the sample (log dilution 100) or log dilutions of the extract at 10 3, 104, 10-5, 10-6 and <br /> 103. Hydrocarbon plates were counted after 7 days incubation at 30°C. The plate count data are <br /> reported as colony forming units (cfu)per gram (g) of soil. Each enumeration value represents a <br /> statistical average of the plate count data obtained from two of the four inoculating log dilutions <br /> assayed. <br /> Protocol for Total Heterotrophic Bacteria: Sterile agar plates (100 x 15 mm) were prepared with <br /> minimal salts medium and 2.35%Difco heterotrophic plate count agar at pH 6.8 without any other <br /> 1 <br />