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1 <br /> love box The late count data are reported as colon forming units (cfu) per milliliter (ml) of <br /> • g p P Y <br /> sample Each enumeration value represents a statistical average of the plate count data obtained <br />' from two of the four inoculating log dilutions assayed <br /> Protocol for Anaerobic Hydrocarbon-Degrading Bacteria Pasteurized Chevron diesel No 2 <br />' and regular gasoline were dissolved into agar plates as the sole carbon and energy sources for the <br /> growth of anaerobic hydrocarbon-degrading bacteria The medium includes alternative terminal <br />' electron acceptors such as sulfate, nitrate, and iron Sterile agar plates (100 x 15 mm) were <br /> prepared with minimal salts medium at pH 6 8 with agar and hydrocarbons, without any other <br /> carbon sources or nutrients added Plates were set up and poured in a Coy anaerobic glove box <br /> under strict anaerobic conditions (atmosphere of nitrogen, carbon dioxide and hydrogen) <br /> Triplicate plates were inoculated with 1 0 ml of each sample (log dilution. 100) or log dilutions of <br /> the sample at 10-t, 10-2, and 10-3 Hydrocarbon plates were counted after 13 days incubation <br />' inside the glove box at ambient temperature The plate count data is reported as colony forming <br /> units (cfu) per milliliter (ml) Each enumeration value represents a statistical average of the plate <br /> count data obtained from two of the four inoculating log dilutions assayed <br /> ANAEROBIC <br />' Hydrocarbon-Degrading and Total Heterotrophic <br /> Bacteria Enumeration Assays <br /> 10 Client Sample Sample SHydrocarbon Target Total <br /> P p Degraders Hydrocarbons Heterotrophs <br /> Number Date cfu/ml Tested cfu/ml <br />' MW-1 12/16/03 4 x 103 Gasoline/Diesel 1 x 104 <br /> MW-4 12/16/03 3 x 102 Gasoline/Diesel 5 x 104 <br />' MW-513 12/16/03 1 x 103 Gasoline/Diesel 3 x 104 <br /> MW-8 12/16/03 2 x 102 Gasoline/Diesel 3 x 102 <br /> Sterile Water 12/17/03 0 Gasoline/Diesel 0 <br /> I Air Control 12/17/03 0 Gasoline/Diesel 0 <br /> Positive Control 12/17/03 4x. 10$ Gasoline/Diesel 8X 10g <br /> fReporting Limit for enumeration data is 1 0 x 10 1 cfu/ml <br /> A positive control sample of hydrocarbon-degrading bacteria was run concurrently with these <br /> samples using a mixed flask culture of bacteria isolated from contaminated groundwater sites in <br /> Northern California <br /> • <br />