Laboratory Maintenance of Nocardia Species

Dipesh Dhakal1, Jae Kyung Sohng1

1 Institute of Biomolecule Reconstruction, Department of BT‐Convergent Pharmaceutical Engineering, Sun Moon University, Chungnam
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10F.1
DOI:  10.1002/9780471729259.mc10f01s39
Online Posting Date:  November, 2015
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Nocardia spp. are aerobic, Gram‐positive, catalase‐positive, non‐motile actinomycetes. Various species of the genus Nocardia have attracted attention due to their detrimental effects on human health. Recent discoveries, however, have exposed their importance as producers of bioactive compounds and degraders of complex organic compounds, as well as their involvement in biotransformation into valuable products. This unit includes general protocols for the laboratory maintenance of Nocardia spp., including growth in liquid medium, growth on solid agar, and long‐term storage. Nocardia sp. CS682 (KCTC11297BP), isolated from soil collected in Jeonnam, Korea, is used as a prototype for explaining the considerations for efficient laboratory maintenance of Nocardia spp. © 2015 by John Wiley & Sons, Inc.

Keywords: Nocardia spp; laboratory growth; glycerol stocks; storage; Nargenicin A1

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Table of Contents

  • Introduction
  • Basic Protocol 1: Growth of Nocardia SP. CS682 on BHI Solid Medium
  • Basic Protocol 2: Growth of Nocardia SP. CS682 in BHI Liquid Medium
  • Basic Protocol 3: Long‐Term Storage of Nocardia SP. CS682
  • Basic Protocol 4: Production of Secondary Metabolite (Nargenicin A1) from Nocardia SP. CS682
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Growth of Nocardia SP. CS682 on BHI Solid Medium

  • Brain‐heart infusion (BHI) solid medium (see recipe)
  • Nocardia sp. CS682 glycerol stock (see protocol 3)
  • Biological safety cabinet or other sterile setting
  • 100 × 15–mm petri dishes, sterile
  • Sterile inoculation needle or wooden stick
  • 37°C incubator

Basic Protocol 2: Growth of Nocardia SP. CS682 in BHI Liquid Medium

  • Brain‐heart infusion (BHI) liquid medium (see recipe)
  • Nocardia sp. CS682 on solid medium (see protocol 1) or glycerol stock (see protocol 3)
  • 250‐ml baffled culture flask, sterile (glass or plastic with vented caps)
  • Toothpicks, sterile
  • Cotton plug, sterile
  • Shaking incubator

Basic Protocol 3: Long‐Term Storage of Nocardia SP. CS682

  • Nocardia sp. CS682 liquid culture (see protocol 2)
  • 30% to 40% (v/v) sterile glycerol (dissolved in water)
  • Biological safety cabinet or other sterile environment
  • 1.5‐ml microcentrifuge tube, sterile
  • 1‐ml pipet, sterile
  • Pipettors
  • −80°C freezer

Basic Protocol 4: Production of Secondary Metabolite (Nargenicin A1) from Nocardia SP. CS682

  • Regenerated Nocardia sp. CS682 culture in brain‐heart infusion (BHI) liquid medium (see protocol 2)
  • Oatmeal broth (see recipe)
  • Ethyl acetate
  • Methanol
  • 500‐ml baffled culture flask, sterile (glass or plastic with vented caps)
  • 1‐ml pipet tips, sterile
  • Micropipettors
  • Shaking incubator (variable temperature)
  • 50‐ml conical centrifuge tube
  • Centrifuge
  • Separatory funnel
  • Rotary evaporator
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Literature Cited

Literature Cited
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