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

J Appl Microbiol. 2003;95(4):677-85. doi: 10.1046/j.1365-2672.2003.02025.x.

Abstract

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.

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Antifungal Agents / pharmacology
  • Centrifugation, Density Gradient / methods*
  • Culture Media
  • DNA, Ribosomal / analysis
  • Nocardia / classification
  • Nocardia / drug effects
  • Nocardia / isolation & purification*
  • Polymorphism, Restriction Fragment Length
  • Soil Microbiology*
  • Spores, Bacterial / drug effects
  • Spores, Bacterial / isolation & purification

Substances

  • Anti-Bacterial Agents
  • Antifungal Agents
  • Culture Media
  • DNA, Ribosomal