Published: July 28th, 2023
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.....
1. Bacteria inoculation
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.......
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.......
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.....
|Cell Density Meter
|Chemical fume hood
|Conincal tubes (50 mL)
|E. coli OP50
|250 mL: FB501250
500 mL: FB501500
1000 mL: FB5011000
|Liquid waste collection bottle
|Magnesium Sulfate (MgSO4)
|Electron Microscopy Sciences
|Paraformaldehyde – methanol free solution
|Eppendorf Easypet 3
|Plastic dishes (100 mm)
|Potassium Phosphate Monobasic (KH2PO4)
|Seahorse XF Calibrant
|Seahorse XFe96 Extracellular Flux Assay Kit and Cell Culture Microplate
|Serological pipettes (50 mL)
|11 676 083
|Sodium Chloride (NaCl)
|Sodium Hydroxide (NaOH)
|Sodium Phosphate Dibasic Anhydrous (Na2HPO4)
|Solid waste collection bucket
|5.0 Gallon M1 Traditional Pail
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