Caenorhabditis elegans as a Model for Toxic Effects of Nanoparticles: Lethality, Growth, and Reproduction

Laura L. Maurer1, Ian T. Ryde1, Xinyu Yang1, Joel N. Meyer1

1 Nicholas School of the Environment, Duke University, Durham, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.10
DOI:  10.1002/0471140856.tx2010s66
Online Posting Date:  November, 2015
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Abstract

The nematode Caenorhabditis elegans is extensively utilized in toxicity studies. C. elegans offers a high degree of homology with higher organisms, and its ease of use and relatively inexpensive maintenance have made it an attractive complement to mammalian and ecotoxicological models. C. elegans provides multiple benefits, including the opportunity to perform relatively high‐throughput assays on whole organisms, a wide range of genetic tools permitting investigation of mechanisms and genetic sensitivity, and transparent bodies that facilitate toxicokinetic studies. This unit describes protocols for three nanotoxicity assays in C. elegans: lethality, growth, and reproduction. This unit focuses on how to use these well‐established assays with nanoparticles, which are being produced in ever‐increasing volume and exhibit physicochemical properties that require alteration of standard toxicity assays. These assays permit a broad phenotypic assessment of nanotoxicity in C. elegans, and, when used in combination with genetic tools and other assays, also permit mechanistic insight. © 2015 by John Wiley & Sons, Inc.

Keywords: Caenorhabditis elegans; nanoparticles; lethality; growth inhibition; reproductive toxicity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Lethality Assay for Nanomaterials in C. elegans
  • Support Protocol 1: Preparation of K Agar Plates Seeded with OP50 Bacteria
  • Support Protocol 2: Synchronizing L1 C. elegans Populations
  • Support Protocol 3: Setup and Use of Copas Biosort Large Particle Flow Cytometer for Worm Dispensing into 96‐Well Plates
  • Support Protocol 4: Manually Dispensing Worms if Copas Biosort is Not Available
  • Basic Protocol 2: Growth Assay for Nanomaterials in C. elegans: Assessment Using Copas Biosort
  • Alternate Protocol 1: Growth Assay for Nanomaterials in C. elegans: Assessment Using Microscopy
  • Support Protocol 5: Preparation of UVC‐Killed UvrA Bacteria
  • Basic Protocol 3: Reproductive Assay for Nanomaterials in C. elegans
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Lethality Assay for Nanomaterials in C. elegans

  Materials
  • K agar plates seeded with OP50 bacteria (see protocol 2)
  • L1‐synchronized young adult C. elegans (see protocol 3)
  • K medium (see recipe)
  • Moderately Hard Reconstituted Water (1× EPA water; see recipe)
  • K agar no‐peptone plates (see recipe)
  • Nanoparticle exposure solution (nanoparticle concentrations and specific nanoparticles used are to be determined by the experimenter; see annotation to step 8)
  • Control particles (Union Biometrica, cat. no. 310‐5071‐001, Union Biometrica) for COPAS quality control
  • 15‐ml tubes (VWR, cat. no. 21008‐216)
  • Centrifuge (Beckman Coulter Allegra 25R)
  • COPAS BioSort large particle flow cytometer (Union Biometrica; see protocol 4 for operation; see protocol 5 for alternative)
  • 96‐well plates (Corning Costar, cat. co. 3596)
  • Light microscope
  • Platinum‐wire worm pick (see annotation to step 10 for instructions on making the worm pick)

Support Protocol 1: Preparation of K Agar Plates Seeded with OP50 Bacteria

  Materials
  • LB broth (see recipe)
  • E. coli (OP50 strain; Caenorhabditis Genetics Center, cat. no. OP50‐1; 10 μl of frozen OP50 stock per 100 ml LB broth)
  • KCl
  • NaCl
  • Bacto‐Peptone (e.g., BD Difco)
  • Bacto‐Agar (e.g., BD Difco)
  • Nystatin solution: dissolve 12.5 mg Nystatin in 10 ml of 100% ethanol
  • 1 M CaCl 2
  • 1 M MgSO 4
  • 10 mg/ml cholesterol (Sigma‐Aldrich, cat. no. C8667) in 100% ethanol (sterile filtered)
  • 70% ethanol
  • 500‐ml glass medium bottle with threaded cap
  • Shaking incubator
  • 4‐liter glass Erlenmeyer flask
  • Magnetic stir plate and stir bar
  • 100 mm × 15 mm cell culture plates, sterile (VWR, cat. no. 25384‐302)
  • Glass rods (stored in 70% ethanol)

Support Protocol 2: Synchronizing L1 C. elegans Populations

  Materials
  • Gravid adult C. elegans (Caenorhabditis Genetics Center; gravid adults are obtained by allowing worms to grow to adulthood where eggs are visible under a light microscope; see step 2)
  • K medium (see recipe)
  • Bleach solution (see recipe)
  • K agar plates seeded with OP50 bacteria (see protocol 2)
  • 15‐ml tubes (VWR, cat. no. 21008‐216)
  • Glass rod (stored in 70% ethanol)
  • Centrifuge (Beckman Coulter Allegra 25R)
  • Orbital shaker
  • 25‐cm2 vented‐cap tissue culture flask (Corning, cat. no. CLS430639)

Support Protocol 3: Setup and Use of Copas Biosort Large Particle Flow Cytometer for Worm Dispensing into 96‐Well Plates

  Materials
  • C. elegans worm population (life stage of worms to be determined by experiment end‐user wishes to perform; Caenorhabditis Genetics Center is an excellent resource for obtaining strains)
  • K medium (see recipe)
  • K agar no‐peptone plates (see recipe)
  • Control particles (Union Biometrica, cat. no. 310‐5071‐001)
  • Moderately Hard Reconstituted Water (1× EPA water; see recipe)
  • Bleach solution (see recipe)
  • 50% bleach: 1:1 bleach (e.g., Clorox): distilled H 2O
  • Cleaning solution (Union Biometrica, cat. no. 300‐5072‐000)
  • 15‐ml tubes (VWR, cat. no. 21008‐216)
  • Centrifuge (Beckman Coulter Allegra 25R)
  • Air compressor (Jun‐Air Item No. 1152000; 115 V, 120PSI)
  • Laser (argon)
  • COPAS BioSort large particle flow cytometer (Union Biometrica)
  • 96‐well plates (Corning Costar, cat. no. 3596)
  • Light microscope

Support Protocol 4: Manually Dispensing Worms if Copas Biosort is Not Available

  Materials
  • Synchronized C. elegans in 25‐cm2 flask (see protocol 3)
  • Moderately Hard Reconstituted Water (1× EPA water; see recipe)
  • 15‐ml tubes (VWR Cat. No. 21008‐216)
  • Centrifuge (Beckman Coulter Allegra 25R)
  • Light microscope
  • Appropriate‐sized multiwell plates

Basic Protocol 2: Growth Assay for Nanomaterials in C. elegans: Assessment Using Copas Biosort

  Materials
  • L1‐synchronized C. elegans (see protocol 3)
  • K agar no‐peptone plates (see recipe)
  • Moderately Hard Reconstituted Water (1× EPA water; see recipe)
  • Sheath fluid (Union Biometrica, cat. no. 300‐5070‐000)
  • Bleach solution (see recipe)
  • Cleaning solution (Union Biometrica, cat. no. 300‐5072‐000)
  • 15‐ml tubes (VWR, cat. no. 21008‐216)
  • Centrifuge (Beckman Coulter Allegra 25R)
  • COPAS BioSort large‐particle flow cytometer (Union Biometrica)
  • 96‐well plates (Corning Costar, cat. no. 3596)
  • Light microscope
  • Additional reagents and equipment for dispensing worms using COPAS BioSort ( protocol 4)

Alternate Protocol 1: Growth Assay for Nanomaterials in C. elegans: Assessment Using Microscopy

  Materials
  • Synchronized L1 C. elegans (see protocol 3)
  • Moderately Hard Reconstituted Water (1× EPA water; see recipe)
  • 2× UVC‐killed UvrA bacteria (see protocol 8)
  • Nanoparticles (see annotation to step 5, below)
  • 12‐well plate
  • Glass microscope slides
  • Glass coverslips
  • NIS Elements software (Nikon)
  • Light microscope with camera (Nikon)
  • Additional reagents and equipment for manually dispensing worms ( protocol 5)

Support Protocol 5: Preparation of UVC‐Killed UvrA Bacteria

  Materials
  • LB broth (see recipe)
  • UvrA strain of E. coli (generous gift from Dr. B. Van Houten; available from the Meyer lab upon request)
  • 500‐ml glass media bottle with threaded cap
  • Inoculant wand (Nichrome wire inoculating loop, Carolina Biological Supply, cat. no. 703060)
  • Incubator with orbital shaker
  • Extra‐large culture plates (VWR, cat. no. 25384‐326)
  • UV lamp
  • Rocker plate
  • 2.0‐ml microcentrifuge tubes

Basic Protocol 3: Reproductive Assay for Nanomaterials in C. elegans

  Materials
  • Synchronized L1 C. elegans (see protocol 3)
  • K agar plates (see recipe) seeded with OP50 bacteria (see protocol 2)
  • Moderately Hard Reconstituted Water (1× EPA water; see recipe)
  • K agar no peptone plates (see recipe)
  • Exposure solution (see annotation to step 8, below)
  • Control particles (Union Biometrica, cat. no. 310‐5071‐001)
  • UVC‐killed UvrA bacteria (see protocol 8)
  • Sheath fluid (Union Biometrica, cat. no. 300‐5070‐000)
  • Bleach solution (see recipe)
  • Cleaning solution (Union Biometrica, cat. no. 300‐5072‐000)
  • 15‐ml tubes (VWR Cat. No. 21008‐216)
  • Centrifuge (Beckman Coulter Allegra 25R)
  • COPAS BioSort large particle flow cytometer (Union Biometrica
  • Computer running Microsoft Excel
  • 96‐well plates (Corning Costar, cat. no. 3596)
  • Additional reagents and equipment for dispensing worms using COPAS BioSort ( protocol 4)
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Figures

Videos

Literature Cited

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