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Application of sucrose-gradient centrifugation for selective isolation of Nocardia spp. from soil
H Yamamura1, M Hayakawa, Y Iimura
1Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University, Kofu, Japan.
Aims:
To devise and evaluate a method for selective isolation of the less abundant actinomycetes, Nocardia spp. in soil.
Methods And Results:
This newly developed method is based on differentiating Nocardia from other actinomycete taxa by centrifugation. A water suspension of air-dried soil is centrifuged through a gradient consisting of 10, 20, 30, 40 and 50% sucrose at 240 x g for 30 min. The 20% sucrose layer, which is enriched with Nocardia spp., is then diluted and plated on humic acid-vitamin agar supplemented with antibacterial agents. The proposed method consistently achieved selective isolation of Nocardia spp. in all 14 soil samples tested, which accounted for 5-89% of the total microbial population recovered. Tentative taxonomic characterization based on a restriction fragment length polymorphism (RFLP) analysis of the 16S ribosomal DNA suggested that many of the soil isolates could belong to N. asteroides, N. salmonicida or N. uniformis.
Conclusions:
Differential centrifugation can successfully and efficiently isolate soil Nocardia populations that are suppressed by conventional dilution plating approaches.
Significance And Impact Of The Study:
The development and application of new methodologies with which to isolate less-explored actinomycete taxa is important for improving our knowledge about their taxonomy, ecology and industrial applications.