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Abstract

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Materials

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Biology

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

Published: July 28th, 2023

DOI:

10.3791/65775

1Molecular and Integrative Physiology Department, University of Michigan, Ann Arbor, 2Department of Internal Medicine, University of Michigan, Ann Arbor
* These authors contributed equally

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.

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.

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.....

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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.......

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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.......

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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.......

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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.

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Name Company Catalog Number Comments
Aluminum Foil Staples 2549291
Bunsen burner VWR 470121-700 
Cell Density Meter Denville 80-3000-45 
Centrifuge Eppendorg 5430
Chemical fume hood Labcono 975050411384RG
Conincal tubes (50 mL) Fisher 339652
Cuvettes  Fisher 14-955-127
E. coli OP50 CGC OP50
Erlenmyer flasks Fisher 250 mL: FB501250
500 mL: FB501500
1000 mL: FB5011000
Inoculation loop Fisher 22-363-605
LB Agar Fisher BP1425500
Liquid waste collection bottle Thomas Scientific 1230G50
Magnesium Sulfate (MgSO4) Sigma M7506
Paraformaldehyde (32%) Electron Microscopy Sciences 15714-S Paraformaldehyde – methanol free solution
Pipettor Eppendorf Eppendorf Easypet 3
Plastic dishes (100 mm) Fisher FB0875712
Potassium Phosphate Monobasic (KH2PO4) Fisher P2853
Seahorse XF Calibrant Agilent 100840-000
Seahorse XFe96 Extracellular Flux Assay Kit and Cell Culture Microplate Agilent 101085-004
Serological pipettes (50 mL) Genesee Scientific 12-107
Shaker incubator Thermo 11 676 083
Sodium Chloride (NaCl) Fisher S640-3
Sodium Hydroxide (NaOH) Fisher S318500
Sodium Phosphate Dibasic Anhydrous (Na2HPO4) Sigma S374-500
Solid waste collection bucket M&M Industries  5.0 Gallon M1 Traditional Pail
Tryptone Genesee Scientific 20-251
Vortex Thermo 11676331
Weighing balance C Goldenwall HZ10K6B
Yeast Extract Genesee Scientific 20-255

  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., Hengartner, M. O. Finding function in novel targets: C. elegans as a model organism. Nature reviews. Drug discovery. 5 (5), 387-398 (2006).
  4. Meneely, P. M., Dahlberg, C. L., Rose, J. K. Working with worms: Caenorhabditis elegans as a model organism. Current Protocols Essential Laboratory Techniques. 19 (1), (2019).
  5. Zhang, S., Li, F., Zhou, T., Wang, G., Li, Z. Caenorhabditis elegans as a useful model for studying aging mutations. Frontiers in Endocrinology. 11, 554994 (2020).
  6. Feng, M., Gao, B., Garcia, L. R., Sun, Q. Microbiota-derived metabolites in regulating the development and physiology of Caenorhabditis elegans. Frontiers in Microbiology. 14, 1035582 (2023).
  7. McIntyre, G., Wright, J., Wong, H. T., Lamendella, R., Chan, J. Effects of FUdR on gene expression in the C. elegans bacterial diet OP50. BMC Research Notes. 14 (1), 207 (2021).
  8. Cabreiro, F., et al. Metformin retards aging in c. Elegans by altering microbial folate and methionine metabolism. Cell. 153 (1), 228-239 (2013).
  9. Worthy, S. E., et al. Identification of attractive odorants released by preferred bacterial food found in the natural habitats of c. Elegans. PLoS One. 13 (7), e0201158 (2018).
  10. Chen, Y. C., Seyedsayamdost, M. R., Ringstad, N. A microbial metabolite synergizes with endogenous serotonin to trigger C. elegans reproductive behavior. Proceedings of the National Academy of Sciences of the United States of America. 117 (48), 30589-30598 (2020).
  11. Kim, D. H., Flavell, S. W. Host-microbe interactions and the behavior of Caenorhabditis elegans. Journal of Neurogenetics. 34 (3-4), 500-509 (2020).
  12. Gems, D., Riddle, D. L. Genetic, behavioral, and environmental determinants of male longevity in Caenorhabditis elegans. Genetics. 154 (4), 1597-1610 (2000).
  13. Qi, B., Kniazeva, M., Han, M. A vitamin-b2-sensing mechanism that regulates gut protease activity to impact animal's food behavior and growth. eLife. 6, e26243 (2017).
  14. Garigan, D., et al. Genetic analysis of tissue aging in caenorhabditis elegans: A role for heat-shock factor and bacterial proliferation. Genetics. 161 (3), 1101-1112 (2002).
  15. Virk, B., et al. Folate acts in E. coli to accelerate C. elegans aging independently of bacterial biosynthesis. Cell Reports. 14 (7), 1611-1620 (2016).
  16. Beydoun, S., et al. An alternative food source for metabolism and longevity studies in Caenorhabditis elegans. Communications Biology. 4 (1), 258 (2021).
  17. Thavarajah, R., Mudimbaimannar, V. K., Elizabeth, J., Rao, U. K., Ranganathan, K. Chemical and physical basics of routine formaldehyde fixation. Journal of Oral and Maxillofacial Pathology. 16 (3), 400-405 (2012).
  18. Felix, H. Permeabilized and immobilized cells. Methods in Enzymology. 137, 637-641 (1988).
  19. Lobritz, M. A., et al. Antibiotic efficacy is linked to bacterial cellular respiration. Proceedings of the National Academy of Sciences of the United States of America. 112 (27), 8173-8180 (2015).
  20. Nadanaciva, S., et al. Assessment of drug-induced mitochondrial dysfunction via altered cellular respiration and acidification measured in a 96-well platform. Journal of Bioenergetics and Biomembranes. 44 (4), 421-437 (2012).
  21. Shtonda, B. B., Avery, L. Dietary choice behavior in Caenorhabditis elegans. The Journal of Experimental biology. 209 (Pt 1), 89-102 (2006).
  22. MacNeil, L. T., Watson, E., Arda, H. E., Zhu, L. J., Walhout, A. J. Diet-induced developmental acceleration independent of tor and insulin in C. elegans. Cell. 153 (1), 240-252 (2013).
  23. Kumar, S., et al. Lifespan extension in C. elegans caused by bacterial colonization of the intestine and subsequent activation of an innate immune response. Developmental Cell. 49 (1), 100-117 (2019).
  24. Nakagawa, H., et al. Effects and mechanisms of prolongevity induced by Lactobacillus gasseri sbt2055 in Caenorhabditis elegans. Aging Cell. 15 (2), 227-236 (2016).
  25. Kaeberlein, T. L., et al. Lifespan extension in Caenorhabditis elegans by complete removal of food. Aging Cell. 5 (6), 487-494 (2006).
  26. Beaudoin-Chabot, C., et al. The unfolded protein response reverses the effects of glucose on lifespan in chemically-sterilized C. elegans. Nature Communication. 13 (1), 5889 (2022).
  27. Komura, T., Takemoto, A., Kosaka, H., Suzuki, T., Nishikawa, Y. Prolonged lifespan, improved perception, and enhanced host defense of Caenorhabditis elegans by Lactococcus cremoris subsp. cremoris.Microbiology Spectrum. 10 (3), e0045421 (2022).
  28. Ye, X., Linton, J. M., Schork, N. J., Buck, L. B., Petrascheck, M. A pharmacological network for lifespan extension in Caenorhabditis elegans. Aging Cell. 13 (2), 206-215 (2014).
  29. Hastings, J., et al. Wormjam: A consensus C. elegans metabolic reconstruction and metabolomics community and workshop series. Worm. 6 (2), e1373939 (2017).
  30. O'Donnell, M. P., Fox, B. W., Chao, P. H., Schroeder, F. C., Sengupta, P. A neurotransmitter produced by gut bacteria modulates host sensory behaviour. Nature. 583 (7816), 415-420 (2020).
  31. Stuhr, N. L., Curran, S. P. Bacterial diets differentially alter lifespan and healthspan trajectories in C. elegans. Communications Biology. 3 (1), 653 (2020).
  32. Dirksen, P., et al. Cembio - the Caenorhabditis elegans microbiome resource. G3 (Bethesda). 10 (9), 3025-3039 (2020).

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