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Isolation of Actinomycetes from Soil ____________________________________ 1.) Obtain a soil sample -Soil type: a) Dry - richest in actinomycetes. b) Neutral to alkaline - Sandy soil favors: Streptomyces - Organic rich soil favors : Microminospora 2.) Create a suspension by mixing 1g of soil with 99 mls of sterile ddH2O.(1E-2 dilution). Take 1 ml of this and dilute it into 9 ml sterile ddH2O (1E-3 dilution). 3.) Heat the suspension (1E-3 dilution) for 16 hours at 45 C. 4.) Plate 0.1 ml and 0.2 ml ( 1E-4, 2E-4 dilution ) onto sodium caseinate plates. 5.) Inxcubate for 5-7 days at room temperature. Many different actinomycetes will appear. A large percentage will probably produce a blue diffusible compound similar in color to that of actinorhodin. Notes: The soil can be pretreated by air drying for 48 hrs. This will decrease the number of non-spore forming bacteria. The addition of antibiotics,such as rifamycin, to richer selection media can also select for actinomycetes. Erythromycin will increase the percentage of Streptomyces species isolated and other antibiotics will have differing effects. Sodium Caseinate Agar Sodium Caseinate 0.20 g/l K2HPO4 0.50 g/l MgSO4 0.20 g/l FeCl3 0.01 g/l Agar 15.0-16.0 g/l pH 6.5-6.7