Myxococcus xanthus Growth, Development, and Isolation

Zalman Vaksman1, Heidi B. Kaplan2

1 Virginia Tech, Blacksburg, Virginia, 2 University of Texas Medical School, Houston, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 7A.1
DOI:  10.1002/9780471729259.mc07a01s39
Online Posting Date:  November, 2015
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Abstract

Myxobacteria are a highly social group among the delta proteobacteria that display unique multicellular behaviors during their complex life cycle and provide a rare opportunity to study the boundary between single cells and multicellularity. These organisms are also unusual as their entire life cycle is surface associated and includes a number of social behaviors: social gliding and rippling motility, ‘wolf‐pack’‐like predation, and self‐organizing complex biostructures, termed fruiting bodies, which are filled with differentiated environmentally resistant spores. Here we present methods for the growth, maintenance, and storage of Myxococcus xanthus, the most commonly studied of the myxobacteria. We also include methods to examine various developmental and social behaviors (fruiting body and spore formation, predation, and rippling motility). As the myxobacteria, similar to the streptomycetes, are excellent sources of many characterized and uncharacterized antibiotics and other natural products, we have provided a protocol for obtaining natural isolates from a variety of environmental sources. © 2015 by John Wiley & Sons, Inc.

Keywords: Mxyococcus xanthus; myxobacteria; microbial development; social behaviors; fruiting body formation; sporulation; predatory bacteria

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Growth of M. xanthus from a Frozen Stock
  • Basic Protocol 2: Maintenance of M. xanthus on Agar Plates
  • Basic Protocol 3: Growth of M. xanthus in Liquid Medium
  • Basic Protocol 4: Resuspension of M. xanthus Liquid‐Grown Cells
  • Basic Protocol 5: Preparation of M. xanthus Frozen Stocks
  • Basic Protocol 6: M. xanthus Fruiting Body Development on Starvation Agar
  • Alternate Protocol 1: M. xanthus Fruiting Body Development in Submerged Culture
  • Basic Protocol 7: Myxospore Isolation and Titer Determination from Starvation Agar Fruiting Bodies
  • Alternate Protocol 2: Myxospore Isolation and Titer Determination from Submerged Culture Fruiting Bodies
  • Basic Protocol 8: Glycerol Spore Generation
  • Basic Protocol 9: M. xanthus Predatory Growth and Rippling Behavior on Starvation Agar
  • Alternate Protocol 3: M. xanthus Predatory Growth and Rippling Behavior on Nutrient Agar
  • Support Protocol 1: Preparation of Prey Cells
  • Basic Protocol 10: Isolating M. xanthus from Environmental Samples
  • Basic Protocol 11: Purifying M. xanthus Isolated from Environmental Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Growth of M. xanthus from a Frozen Stock

  Materials
  • M. xanthus frozen stock ( protocol 5)
  • CTT 1.5% agar plate without antibiotics (see recipe)
  • Wooden applicator stick, sterile
  • Inoculating loop, sterile
  • Plastic shoebox containing wet paper towels
  • Incubator, 32°C (or 30° to 34°C incubator)

Basic Protocol 2: Maintenance of M. xanthus on Agar Plates

  Materials
  • M. xanthus initial CTT streak plate ( protocol 1)
  • CTT 1.5% agar plate with or without antibiotics, as necessary (see recipe)
  • Wooden toothpicks, sterile
  • Plastic shoebox containing wet paper towels
  • Incubator, 32°C (or 30° to 34°C incubator)
  • Parafilm

Basic Protocol 3: Growth of M. xanthus in Liquid Medium

  Materials
  • M. xanthus CTT 1.5% agar plate stock ( protocol 2)
  • CTT broth (see recipe)
  • Antibiotics, as necessary (see recipe)
  • Inoculating loop, sterile
  • Capped 50‐ml Erlenmeyer flask (with or without a side‐arm), sterile
  • Vortex
  • Shaking incubator, 32°C (or 30° to 34°C and air or water incubator)
  • Spectrophotometer or Klett meter

Basic Protocol 4: Resuspension of M. xanthus Liquid‐Grown Cells

  Materials
  • M. xanthus liquid culture at ∼4 × 108 cells/ml ( protocol 3)
  • CTT broth, TPM buffer, or MC7 buffer (see reciperecipes)
  • Capped centrifuge tubes, sterile (10 or 50 ml) or microcentrifuge tubes, sterile
  • Vortex
  • Refrigerated centrifuge or non‐refrigerated microcentrifuge

Basic Protocol 5: Preparation of M. xanthus Frozen Stocks

  Materials
  • M. xanthus liquid culture at ∼4‐5 × 108 cells/ml (see protocol 3)
  • 50% (v/v) glycerol, sterile
  • 2‐ml freezer vials, sterile (Wheaton or Cryovial)
  • Vortex
  • −70°C freezer

Basic Protocol 6: M. xanthus Fruiting Body Development on Starvation Agar

  Materials
  • M. xanthus liquid‐grown cell pellet in centrifuge tube ( protocol 4)
  • TPM buffer (see recipe)
  • TPM starvation 1.5% agar plates (see recipe)
  • Vortex
  • Plastic shoebox containing wet paper towels
  • Incubator, 32°C (or 30° to 34°C incubator)

Alternate Protocol 1: M. xanthus Fruiting Body Development in Submerged Culture

  Materials
  • M. xanthus liquid‐grown cell pellet in centrifuge tube ( protocol 4)
  • MC7 buffer (see recipe)
  • 24‐well polystyrene plate, sterile
  • Vortex
  • Plastic shoebox containing wet paper towels
  • Incubator, 32°C (or 30° to 34°C incubator)

Basic Protocol 7: Myxospore Isolation and Titer Determination from Starvation Agar Fruiting Bodies

  Materials
  • M. xanthus fruiting bodies grown on TPM starvation 1.5% agar ( protocol 6)
  • TPM buffer, sterile (see recipe)
  • CTT 1.5% agar plates (see recipe)
  • Inoculating loop, sterile
  • 1.5‐ml microcentrifuge tubes, sterile
  • Vortex
  • Probe sonicator
  • Water bath at 50°C
  • Plastic shoebox containing wet paper towels
  • Incubator, 32°C (or 30° to 34°C incubator)

Alternate Protocol 2: Myxospore Isolation and Titer Determination from Submerged Culture Fruiting Bodies

  Materials
  • M. xanthus fruiting bodies grown in submerged culture in a 24‐well plate ( protocol 7)
  • MC7 buffer, sterile (see recipe)
  • CTT 1.5% agar plates (see recipe)
  • 1.5‐ml microcentrifuge tubes, sterile
  • Vortex
  • Probe sonicator
  • Water bath at 50°C
  • Plastic shoebox containing wet paper towels
  • Incubator, 32°C (or 30° to 34°C incubator)

Basic Protocol 8: Glycerol Spore Generation

  Materials
  • M. xanthus liquid‐grown cell pellet in centrifuge tube (see protocol 4)
  • CTT broth, sterile (see recipe)
  • CTT containing 1 M glycerol, sterile
  • Vortex
  • Incubator, 32°C (or 30° to 34°C incubator)
  • Petroff‐Hausser counting chamber (or hemocytometer)
  • Phase contrast microscope (with 40× objective lens)

Basic Protocol 9: M. xanthus Predatory Growth and Rippling Behavior on Starvation Agar

  Materials
  • M. xanthus, DK1622 (unlabeled wild type) and MX477 (green fluorescent protein [GFP]‐labeled wild type; see Commentary, Background Information section) liquid‐grown cell pellets in centrifuge tubes ( protocol 4)
  • TPM buffer, sterile (see recipe)
  • TPM starvation 1.5% agar plates, 10 cm or 35 mm diameter (see recipe)
  • Prey cells resuspended in TPM buffer (Support Protocol)
  • 1.5‐ml microcentrifuge tubes, sterile
  • Vortex
  • Incubator, 32°C (or 30° to 34°C incubator)
  • Fluorescence microscope

Alternate Protocol 3: M. xanthus Predatory Growth and Rippling Behavior on Nutrient Agar

  Materials
  • M. xanthus, DK1622 (unlabeled wild type) and MX477 (GFP‐labeled wild type; see Commentary, Background Information section) liquid‐grown cell pellets in centrifuge tubes ( protocol 4)
  • TPM buffer, sterile (see recipe)
  • One‐half CTT 1.5% nutrient agar plates, 10 cm or 35 mm diameter (see recipe)
  • Prey cells grown to mid‐logarithmic phase (Support Protocol)
  • 1.5‐ml microcentrifuge tubes, sterile
  • Vortex
  • Incubator, 32°C (or 30° to 34°C incubator)
  • Fluorescence microscope

Support Protocol 1: Preparation of Prey Cells

  Materials
  • Myxobacteria isolation agar plates (see recipe)
  • Source material:
  • Unsterilized environmental samples (soil, twigs, dung, insect carcasses)
  • Bait material:
  • Sterilized twigs or wood pieces (some soil can be attached), sterilized by autoclaving and dried)
  • Sterilized dung pellets (from wild or domesticated animals that have had no antibiotic exposure), sterilized by autoclaving and dried
  • Plastic shoebox containing wet paper towels
  • Incubator, 32°C (or 30° to 34°C incubator)

Basic Protocol 10: Isolating M. xanthus from Environmental Samples

  Materials
  • Fruiting bodies isolated from environmental samples on myxobacteria isolation agar plates ( protocol 10)
  • Myxobacteria purification agar plates (see recipe)
  • CTT broth (see recipe)
  • TPM starvation 1.5% agar plates (see recipe)
  • CTT 1.5% agar plates without antibiotics (see recipe)
  • 18‐ or 20‐gauge needles
  • Plastic shoebox
  • Humidified incubator, 32°C
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Figures

Videos

Literature Cited

Literature Cited
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