2.6. Enrichment of cultures for isolating hydrocarbon-degrading bacteria

Cultures containing hydrocarbon-degrading bacteria were prepared from samples of surface soil (the upper soil layers) and soil from 20 cm deep (the lower soil layers) from each sampling point and samples of oil waste. A 1 g aliquot of a sample was suspended in 20 mL of Luria Broth (LB). The mixture was then incubated at 30 °C for 72 h on a shaker set to shake at 300 rpm. A 2 mL aliquot of the liquid was then added to 20 mL of mineral salt medium (MSM) supplemented with 1 mL of diesel or crude oil as a carbon source. This adaptation step was repeated three times to ensure that the medium was enriched with bacteria capable of growing using hydrocarbons in crude oil or diesel [19,21,22]. A 100 μL aliquot of an enriched LB liquid culture was then spread on MSM agar, then 100 μL of crude oil or diesel was sprayed onto the agar. Isolates with distinct morphologies were transferred to new LB agar plates and purified by successively sub-culturing the isolated colonies six times. The MSM contained 4.0 g/L NH₄NO₃, 2.0 g/L Na₂HPO₄, 0.53 g/L KH₂PO₄, 0.17 g/L K₂SO₄, 0.10 g/L MgSO₄.7H₂O, 1 g/L ethylenediaminetetraacetic acid, 0.42 g/L ZnSO₄, 1.78 g/L MnSO₄, 0.5 g/L H₃BO₃, and 1 g/L NiCl₂. MSM solid medium was prepared by adding 20 g of agar to 1 L of MSM.