Detection, enumeration and isolation of atrazine-degrading bacteria

Isolation of atrazine-degrading bacteria was commenced immediately after the processing of soil samples. To detect, enumerate and isolate atrazine degraders, 0.1 g of bulk or rhizosphere soil, or (for samples D5 and D6) 0.1 g of common reed or cogon grass root sections with tightly adhering soil were placed into 2 mL polypropylene tubes with 1 mL washing buffer (SM medium salt solution). The tubes were secured in a MO BIO Vortex Adapter assembled on a Vortex-Genie® 2 Vortex (MO BIO Laboratories, Inc., USA) and vortexed at maximum speed for 10 min. Washed root sections from D5 and D6 rhizosphere samples were removed, blotted and weighed to determine the exact amount of soil in the tubes. Serial dilutions of the resulting suspensions were plated onto solid media SM and TY. The plates were incubated at 28 °C. Colonies of soil bacteria growing on TY agar were counted after 5 days. Colonies of atrazine degraders, which produced clearing zones, were first counted after 3-day incubation. The appearance of additional atrazine-degrading colonies was checked daily during the following week. Bacterial densities and confidence intervals were calculated according to Koch [59].

For isolation of atrazine degraders, colonies were chosen based on their morphology, size of clearing zones and time required for their production. Several colonies within each distinguishable type were selected to ensure the most complete isolation of different bacteria. The colonies were repeatedly streaked on SMY agar until cultures of atrazine-degrading bacteria showing no presence of contaminating microorganisms on SMY, TY and R2A (Sigma-Aldrich, USA) agar media were obtained.