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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The food source for Caenorhabditis elegans in the lab is live Escherichia coli. Since bacteria are metabolically active, they present a confounding variable in metabolic and drug studies in C. elegans. A detailed protocol to metabolically inactivate bacteria using paraformaldehyde is described here.

Abstract

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a diet of live bacteria, the metabolic activity of their food source can confound experiments looking for the direct effects of various interventions on the worm. To avoid the confounding effects of bacterial metabolism, C. elegans researchers have used multiple methods to metabolically inactivate bacteria, including ultraviolet (UV)-irradiation, heat-killing, and antibiotics. UV treatment is relatively low-throughput and cannot be used in liquid culture because each plate must be examined for successful bacterial killing. A second treatment method, heat-killing, negatively affects the texture and nutritional quality of the bacteria, leading to the developmental arrest of C. elegans. Finally, antibiotic treatment can directly alter C. elegans physiology in addition to preventing bacterial growth. This manuscript describes an alternative method to metabolically inactivate bacteria using paraformaldehyde (PFA). PFA treatment cross-links proteins within bacterial cells to prevent metabolic activity while preserving cellular structure and nutritional content. This method is high-throughput and can be used in liquid culture or solid plates, as testing one plate of PFA-treated bacteria for growth validates the whole batch. Metabolic inactivation through PFA treatment can be used to eliminate the confounding effects of bacterial metabolism on studies of drug or metabolite supplementation, stress resistance, metabolomics, and behavior in C. elegans.

Introduction

Caenorhabditis elegans was originally proposed as a model organism in 19651 and has since been widely adopted in studies of genetics, development, behavior, aging, and metabolism2. Due to their large brood size and transparent cuticle, C. elegans is particularly well-suited for high-throughput screening with fluorescent reporters3. Their short life cycle, hermaphroditic reproduction, and genetic homology with humans also make C. elegans a valuable model system for studies on development4 and aging biology5. Moreover, C.....

Protocol

1. Bacteria inoculation

  1. Prepare Luria broth (LB) by dissolving 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride (NaCl) in 950 mL of distilled water.
  2. Adjust the pH of the LB to 7.0 by adding 5M sodium hydroxide (NaOH). This should only require about 0.2 mL of NaOH.
  3. Autoclave the pH-adjusted LB media on a liquid cycle for 45 min at 15 psi. Allow the solution to cool and store at room temperature.
  4. Inoculate a single colony of bacteria in 100 mL of.......

Representative Results

A detailed workflow of the protocol is shown in Figure 1. A high-throughput method was developed and optimized to consistently inactivate bacterial replication (Figure 2A) and metabolism (Figure 2B) for metabolic and drug studies in C. elegans research using paraformaldehyde16. The goal was to determine the lowest concentration of PFA needed and the shortest amount of time required to consistently ki.......

Discussion

Benefits of PFA-killing relative to other bacterial-killing methods
PFA-treatment is a high-throughput method to prevent bacterial metabolism while maintaining a nutritious food source for C. elegans. Killing bacteria via PFA-treatment has multiple advantages over other methods. Unlike UV-treatment, where every plate must be tested for successful killing, a single plate from a batch of PFA-treated bacteria can be tested to validate the batch16. PFA-treatment is also.......

Acknowledgements

This work was funded by NIH R21AG059117 and the Paul F. Glenn Laboratories for Biology of Aging Research at the University of Michigan. SB was funded by T32AG000114. ESK was funded by NSF DGE 1841052.

....

Materials

NameCompanyCatalog NumberComments
Aluminum FoilStaples2549291
Bunsen burnerVWR470121-700 
Cell Density MeterDenville80-3000-45 
CentrifugeEppendorg5430
Chemical fume hoodLabcono975050411384RG
Conincal tubes (50 mL)Fisher339652
Cuvettes Fisher14-955-127
E. coli OP50CGCOP50
Erlenmyer flasksFisher250 mL: FB501250
500 mL: FB501500
1000 mL: FB5011000
Inoculation loopFisher22-363-605
LB AgarFisherBP1425500
Liquid waste collection bottleThomas Scientific1230G50
Magnesium Sulfate (MgSO4)SigmaM7506
Paraformaldehyde (32%)Electron Microscopy Sciences15714-SParaformaldehyde – methanol free solution
PipettorEppendorfEppendorf Easypet 3
Plastic dishes (100 mm)FisherFB0875712
Potassium Phosphate Monobasic (KH2PO4)FisherP2853
Seahorse XF CalibrantAgilent100840-000
Seahorse XFe96 Extracellular Flux Assay Kit and Cell Culture MicroplateAgilent101085-004
Serological pipettes (50 mL)Genesee Scientific12-107
Shaker incubatorThermo11 676 083
Sodium Chloride (NaCl)FisherS640-3
Sodium Hydroxide (NaOH)FisherS318500
Sodium Phosphate Dibasic Anhydrous (Na2HPO4)SigmaS374-500
Solid waste collection bucketM&M Industries 5.0 Gallon M1 Traditional Pail
TryptoneGenesee Scientific20-251
VortexThermo11676331
Weighing balanceC GoldenwallHZ10K6B
Yeast ExtractGenesee Scientific20-255

References

  1. Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. C. . Elegans II. 33, (1997).
  2. Corsi, A. K., Wightman, B., Chalfie, M. A transparent window into biology: A primer on caenorhabditis elegans. WormBook. , 1-31 (2015).
  3. Kaletta, T....

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Caenorhabditis ElegansBacterial DietMetabolic InactivationParaformaldehydeUV irradiationHeat killingAntibioticsBacterial MetabolismHigh throughputLiquid CultureSolid PlatesDrug metabolite SupplementationStress ResistanceMetabolomicsBehavior

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