3.2. Isolation of Actinomycetes

Isolation and enumeration of actinomycetes were done using a serial dilution of Humic Acid-Vitamin (HV) medium [48] and/or NBRC No. 802 Medium [49] by using the direct method [50], the dry heat method [51], and the phenol method [51]. In the direct method, an air-dried soil sample or marine sediment was ground in a mortar and heated in a hot-air oven at 110 °C for 30 min. One gram of the heated samples was transferred to 10 mL of sterile water and mixed for 2 min, then diluted with sterile water to 10⁻¹, 10⁻², and 10⁻³ times. In total, 200 µL of each dilution was inoculated on isolation medium agar of HV [48] or NBRC No. 802 Medium [49] with or without the addition of 1% NaCl. The inoculated plates were incubated for 2–4 weeks at 28 °C. The colonies showing the Streptomyces morphological characteristics were selected and streaked on fresh plates of the modified Streptomyces International Project 2 (ISP2 ≙ YM) agar [52]. The cultures were resuspended in sterile 0.9% (w/v) saline supplemented with 15% (v/v) glycerol and stored at −80 °C. This dry-heat method [51] was used to isolate heat-tolerant actinomycetes spores. In the dry-heat method, the soil or sediment samples were incubated at 100 °C for 40 min and then cooled to 28 °C in a desiccator. The samples were distributed on HV medium agar plates with a spatula tip and incubated at 28 °C for 2–3 weeks. The phenol method was used to select for spores, which survive in the presence of phenol. In total, 1 mL of 10⁻¹ dilution of one gram of oven-dried soil or marine sample was transferred to 9 mL of sterile 5 mM-phosphate buffer (pH 7.0) containing phenol at a final concentration of 1.5%. The sample was then heated and diluted in serial dilution (10⁻¹, 10⁻², 10⁻³). Next, 100 or 200 µL of each dilution was spread over the surface of HV medium agar plates and incubated for 2–4 weeks at 28 °C.