NEW METHODS FOR THE HIGHLY SELECTIVE ISOLATION OF MICROMONOSPORA AND MICROBISPORA FROM SOIL

Two plate culture methods for the highly selective isolation of the soil actinomycetes Micromonospora and Microbispora, are described, which utilize the differential antibiotic resistance of these microbes and the superior ability of their spores to withstand deleterious agents. (i) To isolate micromonosporae, a water suspension of air-dried soil is first treated with 1.5% phenol, diluted with water and then cultured on HV agar supplemented with both tunicamycin and nalidixic acid. (ii) To isolate microbisporae, air-dried soil is heated in a hot-air oven at 120-degrees-C for 1 h. A water suspension of the heated sample is then treated with a solution of 1.5% phenol and 0.03% chlorhexidine gluconate (CG), diluted and cultured on HV agar supplemented with nalidixic acid. The phenol pretreatment of the soil killed bacteria and streptomycetes in the samples, while keeping micromonosporae and microbisporae alive. The use of tunicamycin suppressed the growth of the remaining microbisporae on HV agar isolation plates, thereby facilitating the intensive isolation of micromonosporae. On the other hand, the dry heat treatment of the soil drastically reduced the number of bacteria in the sample and significantly reduced the numbers of streptomycetes and micromonosporae, without reducing the microbisporae content. Further treatment with a mixture of phenol and CG eliminated dry heat-resisting actinomycetes apart from microbisporae. Dilution plating of samples subjected to heat and the phenol-CG treatment resulted in the intensive isolation of microbisporae on HV agar. From various field soils (vegetable, orchard, corn, and rice paddy), the methods described above always enabled the highly selective isolation of micromonosporae and microbisporae, respectively, as each group accounted for more than 90% of the total number of colonies recovered.