Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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. elegans are relatively easy to maintain. Worms can be grown in liquid culture or on solid agar plates and consume a diet of live Escherichia coli OP50 bacteria⁴.

However, the live food source of C. elegans can confound studies of metabolism, drug supplementation, and behavior. Because live bacteria have their own metabolism, experimental conditions that affect the bacteria also alter the nutrients and metabolites available to the worms. For example, differences in bacterial iron, amino acid, and folate concentrations have diverse effects on C. elegans' development, physiology, and lifespan⁶. Many common lab practices can elicit such changes in the nutrient composition and metabolites produced by OP50. Specifically, exposure to 5-fluoro-2'-deoxyuridine (FUdR), a compound commonly used to prevent reproduction in C. elegans, elicits broad changes in OP50 gene expression, including amino acid biosynthesis pathways⁷. Live bacteria can also confound studies in which C. elegans are supplemented with small molecules because bacteria can partially or completely metabolize the active compounds. Moreover, the effects of these small molecules on the bacteria can, in turn, alter C. elegans physiology, as was reported with the lifespan-extending drug metformin⁸. Finally, live bacteria can change the worm's environment in ways that alter behavior, such as secreting attractive odorants⁹, producing exogenous neuromodulators¹⁰, and creating oxygen gradients in a dense bacteria lawn¹¹.

To mitigate the confounding effects of bacterial metabolism on C. elegans research, multiple methods for killing bacteria have been developed (Table 1). Three common strategies for killing OP50 are UV-irradiation, heat-killing, and antibiotic treatment. While straightforward and relatively low-cost, each of these methods can have undesirable effects on both bacteria and C. elegans. UV-killing via a UV crosslinker¹² is low-throughput and the rate is limited by the number of plates that can fit in the UV crosslinker. Moreover, the efficacy of UV-killing can vary from plate to plate within a batch, and testing for growth on all plates can become difficult in large experiments. Heat-killing OP50 by exposing culture to temperatures of >60 °C comes with a separate set of challenges. High heat can damage nutrients essential for the worm and destroy the cellular structure of bacteria, creating a softer texture that decreases the amount of time worms spend on the food¹³. This method also cannot be used throughout the life cycle of C. elegans because worms fed heat-killed bacteria can arrest early in development¹³. Antibiotic treatment is a third common method for suppressing bacterial metabolism¹⁴, but antibiotics can also alter worm growth and metabolism¹⁵.

One solution to eliminate the metabolic effects of live bacteria while preserving bacterial structure and essential nutrients is to kill OP50 with paraformaldehyde (PFA)¹⁶. PFA is a polymer of formaldehyde that can crosslink proteins within cells¹⁷ to prevent bacterial replication without destroying internal cell structures like the inner plasma membrane¹⁸. Due to this preservation of internal cellular structure, PFA-treated bacteria exhibit no growth or metabolic activity but remain an edible and nutrient-rich food source for C. elegans¹⁶. Here, a detailed protocol is provided which shows how to metabolically inactivate bacteria using paraformaldehyde.

Method	Required Materials	Scalable?		Nutritional?	Effects on Worm?
UV	UV-crosslinker	Limited by:		Yes	Variable effects on lifespan on NGM12, 23, 24
		Number of plates that fit in UV-crosslinker			Variable effects on lifespan on FUdR24, 26, 27
		Irradiation time per plate			Decreased food preference16
		Ability to check every plate for growth8
Heat	>60 °C incubator	Yes		No: destroys cell wall, decreased nutritional value	Developmental arrest 13
					Decreased food preference13
					Extends lifespan on NGM31
Antibiotics	Antibiotics (kanamycin, carbenicillin, etc.)	Yes		Yes	Delays growth and development15
					Extends lifespan in liquid media19
					Extends lifespan on NGM15
PFA	0.5% Paraformaldehyde	Yes		Yes	Small brood size decrease16
					Small development time increase16
					Decreased food preference16

Table 1. Comparisons of methods to kill OP50. UV-killing, heat-killing, antibiotic-treatment, and PFA-treatment have varied effects on the nutritional status of the bacteria and the health of worms fed treated bacteria. These methods for replicatively inactivating E. coli also differ in their required materials and scalability.

Protokół

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 LB in a 500 mL Erlenmeyer flask. Culture the bacteria overnight in a 37 °C shaker incubator.
5. Depending on the health of the bacterial colony, the size of the flask and the speed that the shaker is set to, the time that it takes for the bacteria to grow may vary. After ~14 h, check the optical density (OD) of the bacteria at 600 nm (OD₆₀₀).
6. Remove the bacteria from the shaker when the OD₆₀₀ is 3.0 (1 x 10⁹ colony forming units (CFU)/mL). If the OD₆₀₀ is less than 3.0, return the flask to the shaker incubator until the desired OD is reached.
7. Aliquot the bacteria in 50 mL conical tubes and store at 4 °C or proceed to the next step.

2. Working with paraformaldehyde

NOTE: The concentration of paraformaldehyde (PFA) used, and the duration of exposure may vary somewhat depending on climate, location, and type of bacteria being treated. A good starting point for OP50 is exposure to 0.5% PFA for 1 h, whereas 0.25% PFA for 1 h may be sufficient for HT115.

1. Prepare 32% PFA stock or use commercially purchased 32% PFA solution. Use proper personal protective equipment (PPE) when working with PFA. Wear gloves and eye protection.
2. Add PFA inside a chemical fume hood with proper ventilation. Dispose of PFA containing washes and tips in proper chemical hazard containers in the fume hood.

3. Bacterial treatment with paraformaldehyde

1. Once the bacteria reach an OD₆₀₀ of 3.0, use a serological pipette to transfer 50 mL into a new 250 mL Erlenmeyer flask. Save the rest for a live control, a mock-treated control (see step 4), or to treat as well with PFA as needed.
2. Avoid pouring from one flask to another and be cautious not to splash the sides of the new flask with bacteria. Colonies on the side of the flask may receive a lower dose of PFA.
3. In the chemical hood, add 781 µL of 32% PFA to 50 mL bacteria to bring the final concentration to 0.5%. Dispose of the tip used in a solid waste chemical hazard container.
4. Cover the flask with foil and return to the 37 °C shaker incubator for 1 h. After 1 h, remove the flask from the incubator and proceed to step 5.

4. Mock-treated control

1. Once the bacteria reach an OD₆₀₀ of 3.0, use a serological pipette to transfer 50 mL of the bacteria to a 50 mL conical tube.
2. Proceed to step 5.3 to complete the washing steps similar to the PFA-treated group.

5. Washing the bacteria to remove residual PFA

1. In the chemical hood, use a serological pipette to transfer the treated bacteria from the Erlenmeyer flask to a 50 mL conical tube. Using a serological pipette instead of pouring the bacteria will prevent contamination from any bacterial colonies on the edge of the flask that may have avoided direct treatment with PFA.
2. Centrifuge treated bacteria at approximately 3000 x g for 20 min. Remove the supernatant by disposing of it in a liquid waste chemical hazard container in the chemical hood.
3. Add 25 mL of LB and vortex to resuspend the bacterial pellet (Filling the tube fully makes it harder to resuspend the pellet). Repeat centrifugation and pellet resuspension 4x.
4. Resuspend the pellet in volumes optimal for different assays. For lifespan assays, seed 60 mm plates with 200 µL of bacteria resuspended in 10 mL of LB. Resuspending the bacteria in 10 mL of LB results in a 5x concentration from the original 50 mL culture.
5. Store the bacteria at 4 °C.

6. Quality check of bacterial growth

1. After the final wash and resuspension, streak an LB plate (using a sterile pipette tip) with the prepared bacteria. It is good practice to streak the LB used for washing and resuspending on a separate plate as well to make sure that the LB used was not contaminated.
2. Place the plates in a 37 °C incubator overnight. Check for any growth. The bacteria are considered replicatively dead when colonies do not grow on the LB plate.

7. Quality check for bacterial metabolism using a respirometer

1. After the final wash and resuspension from step 5.4, confirm that the bacteria are metabolically dead using available tools such as respirometers^19,20 and measuring the basal oxygen consumption rate (OCR).
2. Prepare M9 solution: Dissolve 3 g of potassium phosphate monobasic (KH₂PO₄), 6 g of sodium phosphate dibasic (Na₂HPO₄), and 5 g of sodium chloride (NaCl) in 950 mL of distilled water. Autoclave on a liquid cycle for 45 min at 15 psi, then allow the solution to cool to room temperature. Add 1 mL of 1 M magnesium sulfate (MgSO₄) and store at room temperature.
3. Hydrate the respirometer cartridge: Add 200 µL of calibrant to all wells of a 96-well plate. Place the cartridge in the 96-well plate and incubate overnight in a 37 °C incubator.
4. Assay calibration: On the following day, place the hydrated cartridge in the machine and begin calibration.
5. Assay test plate setup: Using a new 96-well plate, add 160 µL of M9 and 40 µL of the prepped bacteria (1 x 10⁹ CFU/mL) to test wells. Add 200 µL of M9 to the 4 corner wells to use as blank wells. Add 160 µL of M9 and 40 µL of LB used for washing and resuspending to use as negative controls. Add 200 µL of M9 to the rest of the wells that will not be used.
6. Run the assay: Once the cartridge calibration is complete, insert the assay plate from step 7.5 into the machine for analysis. The settings include steps to mix, wait, measure and loop. The results will be shown as oxygen consumption rate (OCR). The bacteria are metabolically dead and ready to use when the OCR is zero.

Figure 1. Workflow for paraformaldehyde treatment. A single colony of E. coli OP50 bacteria is grown overnight. PFA is added to a final concentration of 0.5%, and the PFA-treated culture is shaken for 1 h at 37 °C. Finally, the PFA is removed by washing the culture with fresh LB 5x. To confirm the treated bacteria are replicateively inactive, streak out an LB plate of the treated bacteria and grow overnight. Please click here to view a larger version of this figure.

Wyniki

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...

Dyskusje

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...

Materiały

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