The Nematode C. elegans as an Animal Model to Explore Toxicology In Vivo: Solid and Axenic Growth Culture Conditions and Compound Exposure Parameters

Richard Nass1, Iqbal Hamza2

1 Vanderbilt University Medical Center, Nashville, Tennessee, 2 University of Maryland, College Park, Maryland
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 1.9
DOI:  10.1002/0471140856.tx0109s31
Online Posting Date:  February, 2007
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Abstract

Significant limitations in vertebrate animal model systems include the time involved, the expense, the fact that in vitro results may not reflect live animal pathology, difficulties in transporting the toxin past the blood brain barrier, and the inability to identify the mechanism of action without some a priori knowledge of the toxin's target. The availability of the complete genome sequence of the nematode C. elegans, coupled with the worm's size, growth rate, ease of culturing, and the realization that basic biological mechanisms and disease processes between worms and humans are highly conserved, makes this genetically tractable model a remarkable opportunity to dissect and identify in vivo the cellular processes involved in toxin‐induced cell dysregulation and death. This unit includes protocols for culturing worms on solid and axenic media and acute and chronic exposure parameters for Parkinson's disease–associated toxins and hemin chloride. These methods provide the groundwork for using this powerful model system to further elucidate and understand the molecular mechanisms involved in nutrition as well as toxicological responses relevant to human diseases.

Keywords: liquid media; 6‐OHDA; Parkinson's Disease; high‐throughput; metal; nutrition

     
 
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Table of Contents

  • Basic Protocol 1: Growth, Maintanance, and Assay of C. elegans for Acute and Chronic Exposure to Neurotoxins on Solid Media
  • Alternate Protocol 1: Chronic Exposure of C. elegans to Copper on Agar in Microtiter Plates
  • Basic Protocol 2: Maintenance of C. elegans in Liquid Axenic Medium for Biochemical Studies
  • Alternate Protocol 2: Growing C. elegans in Reduced Metal Medium (mCeHR‐2)
  • Alternate Protocol 3: Growing C. elegans in Metal‐Chelated Medium (mCeHR‐3)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Growth, Maintanance, and Assay of C. elegans for Acute and Chronic Exposure to Neurotoxins on Solid Media

  Materials
  • NaCl
  • Bactoagar (Becton Dickinson)
  • Bactopeptone (Becton Dickinson)
  • 0.5 M potassium phosphate buffer, pH 6.0 ( appendix 2A)
  • 1 M MgSO 4
  • 1 M CaCl 2
  • Cholesterol solution (see recipe)
  • NA22 bacteria (see recipe)
  • Streptomycin
  • Nystatin solution (see recipe)
  • OP50 bacteria culture (see recipe)
  • Synchronization solution (see recipe)
  • M9 buffer (see recipe)
  • 6‐OHDA stock solution (see recipe)
  • Agar pad solution (2% w/v agar)
  • 2% (w/v) sodium azide
  • C. elegans (Bristol N2, fluorescently labeled, or other strain)
  • 2‐liter autoclavable bottles (containing a stir bar) with caps
  • 90‐mm plastic bacterial petri dishes
  • Bent glass rod or Pasteur pipet (“hockey stick”) for spreading culture
  • Laminar flow hood, optional
  • 60‐mm petri dishes or 24‐well plates
  • 15‐ml conical tubes, sterile
  • Beckman CS‐6R centrifuge with GH3.7 horizontal rotor (or equivalent centrifuge or rotor)
  • Phase‐contrast or fluorescence microscope (inverted, confocal, or dissecting)
  • Nutator (Becton Dickinson)
  • 1.6‐ml silicon‐coated microcentrifuge tube
  • 90‐mm NGM agar plate
  • 20°C refrigerated incubator with platform rockers
  • Pasteur pipet or a worm pick
  • Stereo fluorescence microscope with bottom illumination (e.g., Leica MZ16 FA)

Alternate Protocol 1: Chronic Exposure of C. elegans to Copper on Agar in Microtiter Plates

  • 300 mg/liter CuCl 2
  • L1 worms (see protocol 1)

Basic Protocol 2: Maintenance of C. elegans in Liquid Axenic Medium for Biochemical Studies

  Materials
  • C. elegans (Bristol N2, fluorescently labeled, or other strain)
  • 60‐mm NGM agar plates spotted with OP50 E. coli strain (see protocol 1, steps 6 to 10)
  • M9 buffer (see recipe)
  • 0.1 N NaCl
  • mCeHR growth medium (see recipe), supplemented with 100 µg/ml tetracycline
  • 5 N NaOH
  • 5% (v/v) bleach
  • mCeHR growth medium (see recipe)
  • S buffer (see recipe)
  • Freezing solution (see recipe)
  • 5 mM and 10 mM sodium azide (NaN 3)
  • 0.3 M NH 4OH, pH 8.0
  • Hemin chloride
  • 15‐ and 50‐ml conical tubes, sterile
  • 25‐ or 75‐cm2 tissue culture flasks (Nunc)
  • 20°C refrigerated incubator equipped with platform shaker or rocker (Labline Orbit Shaker or Hoefer Rocker)
  • Beckman CS‐6R centrifuge with GH3.7 horizontal rotor (or equivalent rotor)
  • Vortex Genie‐2
  • Phase‐contrast inverted microscope with 10× objective
  • 2‐ml screw cap cryostat freezing vials
  • Glass slides
  • 24‐well microtiter plate

Alternate Protocol 2: Growing C. elegans in Reduced Metal Medium (mCeHR‐2)

  • 50× chelation mix
  • Lactalbumin (see recipe)
  • UHT skim milk, sterile (see recipe)
  • 1‐kDa MWCO dialysis tubing
  • 0.2‐µm filter
  • Additional reagents and equipment for dialysis ( appendix 3H)
NOTE: Wash all glassware with 6 N HCl followed by copious rinsing with distilled deionized water to remove any contaminating metals bound to the glassware.
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Figures

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Literature Cited

Literature Cited
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