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cyto <br /> 7ufture <br /> ENVIRONMENTAL <br /> BIOTECHNOLOGY MAR 2 1 2003 <br /> CytoCulture International,Inc <br /> 249 Tewksbury Avenue -- T <br /> Pt Richmond,CA 94801 USA <br /> Clearwater Group Reporting Date: March 20, 2003 <br /> Protect Name: Barnes Trucking CytoCulture Lab Login: 03-35 <br /> Project Manager: Brian Pierskalla Project number: ZB178C <br /> Address: 229 Tewksbury Ave Tel: 510-307-9943 Fax: 510-232-2823 <br /> Point Richmond, CA 94801 <br /> Samples: Three water samples on ice were received 03/06/03 They were stored at 4°C <br /> and assayed on the same day Please see attached chain of custody <br /> AEROBIC <br /> Hydrocarbon-Degrading and Total Heterotrophic Bacteria Enumeration Assays <br /> Analysis Request Enumeration of aerobic petroleum hydrocarbon-degrading bacteria <br /> (broad range petroleum derived from gasoline and diesel) and aerobic total heterotrophic <br /> bacteria by method 9215A (HPC)/ Standard Methods 9215B modified <br /> Carbon Source: Pasteurized Chevron gasoline No 2 and diesel were dissolved into agar <br /> plates as the sole carbon and energy source for the growth of aerobic hydrocarbon- <br /> degrading bacteria <br /> Protocol for Hydrocarbon-Degrading Bacteria: Sterile agar plates (100x 15 mm) were <br /> prepared with minimal salts medium at pH 6 8 with agar and hydrocarbons, without any <br /> other carbon sources or nutrients added Triplicate plates were inoculated with 10 ml of <br /> each sample (log dilution 10°) or log dilutions of each sample at 10-1, 10'2, and 10-3 <br /> Hydrocarbon plates were counted after 7 days incubation at 30 degrees Celsius The <br /> plate count data is reported as colony forming units (cfu) per milliliter (ml) Each <br /> enumeration value represents a statistical average of the plate count data obtained from <br /> two of the four inoculating log dilutions assayed <br /> Protocol for Total Heterotrophic Bacteria: Sterile agar plates (100 x 15 mm) were <br /> prepared with minimal salts and 2 35%heterotrophic plate count agar at pH 6 8 without <br /> any other carbon source or nutrients added Triplicate plates were inoculated with 1 0 ml <br /> of each sample at log dilutions 10-i, 10-2, and 10 3 and 104 The heterotrophic plates <br /> were counted after 3 days of incubation at 30 Deg C The plate count data is reported as <br /> colony forming units (cfu) per milliliter (ml) of sample Each enumeration value <br /> represents a statistical average of two of the four inoculating log dilutions assayed <br /> • <br />