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AEROBIC <br /> Hydrocarbon-Degrading and Total Heterotrophic Bacteria Enumeration Assay <br /> Results <br /> Client Sample Sample Hydrocarbon Target Total <br /> Number Date Degraders Hydrocarbons Heterotrophs <br /> (cfu/ml) Tested (cfu/ml) <br /> MW-1 03/05/03 l x 104 Gasoline/Diesel 1 5 x 104 <br /> MW-4 03/05/03 3 x 104 Gasoline/Diesel 3 x 104 <br /> MW-5B 03/05/03 1 5x 104 Gasoline/Diesel 8 x 105 <br /> Sterile Water 03/06/03 Zero Gasoline/Diesel Zero <br /> Air Control 03/06/03 Zero Gasoline/Diesel Zero <br /> Positive Control 03/05/03 3 x 107 Gasoline/Diesel 7 x 108 <br /> Reporting Limit for enumeration data is 1 0 x 101 cfu/m1 <br /> A positive control sample of hydrocarbon-degrading bacteria was run concurrently with <br /> these samples using a mixed flask culture of bacteria isolated from contaminated <br /> groundwater sites in Northern California <br /> ANAEROBIC <br /> Hydrocarbon-Degrading Bacteria Enumeration Assay <br /> Analysis Request. Enumeration of anaerobic petroleum hydrocarbon-degrading bacteria <br /> (broad range petroleum hydrocarbons derived from diesel and gasoline) by method <br /> 9215A (HPC) / Standard Methods 9215B modified for anaerobic conditions <br /> Carbon Source: Pasteurized Chevron No 2 diesel and gasoline were dissolved into agar <br /> plates as the sole carbon and energy sources for the growth of hydrocarbon-degrading <br /> anaerobic bacteria The medium includes alternative terminal electron acceptors such as <br /> sulfate, nitrate, and iron <br /> Protocol for Anaerobic Hydrocarbon-Degrading Bacteria. Sterile agar plates (100 x <br /> 15 mm) were prepared with minimal salts medium at pH 6 8 with agar and hydrocarbons, <br /> without any other carbon sources or nutrients added Plates were setup and poured in a <br /> Coy anaerobic glove box under strict anaerobic conditions (atmosphere of nitrogen, <br /> carbon dioxide and hydrogen) Triplicate plates were inoculated with 1 0 ml of each <br /> sample (log dilution 10°) or log dilutions of the sample at 10-i, 10 2,and 10-3 <br /> Hydrocarbon plates were counted after 16 days incubation in the glove box at ambient <br /> temperature The plate count data is reported as colony forming units (cfu) per milliliter <br /> (ml) Each enumeration value represents a statistical average of two of the four <br /> inoculating log dilutions assayed <br />