IngentaConnect Application of sucrose-gradient centrifugation for selective isol...

Abstract:

h. yamamura, m. hayakawa and y. iimura. 2003. 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 × 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.

Keywords: 16S rDNA; differential centrifugation; Nocardia; RFLP; selective isolation; soil bacteria

Document Type: Research article

DOI: 10.1046/j.1365-2672.2003.02025.x

Affiliations: 1: Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University, Kofu, Japan

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Application of sucrose-gradient centrifugation for selective isolation of Nocardia spp. from soil