Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

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. 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.

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 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.

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 kill the bacteria without impacting various measures of health in the worms. These values may vary from one location to another depending on lab environment, and from one bacterial strain to another. For example, exposure of HB101 bacteria to 0.25% for 1 h is sufficient to render it metabolically inactive (Figure 2C), while Enterococcus faecalis (EF) requires a 1.0% PFA concentration (Figure 2D) for a consistent effect. An approach for individual lab optimization was previously established¹⁶ and is detailed in this current work.

Food attraction and consumption
As bacteria grow and replicate, they release various metabolites and pheromones that are attractive to the worms. To determine whether the worms remain attracted to the PFA-treated OP50, plates were seeded with PFA-treated or mock-treated OP50 and the percentage of worms present on the food lawn as compared to off the lawn was tabulated. Data show that worms are attracted to the PFA-treated OP50, since most of the worms remain on the bacterial lawn after 1 h (Figure 3A), similar to what is observed with the mock-treated control (Figure 3B). However, as expected, a sensitive pairwise assay²¹ shows that C. elegans have a stronger preference for the mock-treated live control (Figure 3C) when seeded with the PFA-treated on the same plate. Having established that worms are attracted to PFA-killed bacteria, albeit less than live bacteria, it was imperative to establish that they eat the PFA-killed food. To determine whether the worms consume the PFA-treated OP50, the pumping rate of the worms on different food types was measured. As shown in Figure 3D, worms on PFA-treated OP50 have a higher pumping rate (+25%) than worms on the mock-treated control. This indicates that worms are not purposefully slowing their rate of eating on PFA-killed food and could either indicate a higher rate of eating or a compensatory response to a change in the ease of pumping the treated food.

Fecundity, development, and lifespan
Changes in food conditions can have physiological effects on worms^13,22. To test for broad effects, changes in the rate of development, fecundity, and lifespan were measured. PFA-treated OP50 slightly delays development time (~4 h) of wildtype N2 worms as shown in Figure 4A. Monitoring the egg-laying capacity of worms grown on different bacterial conditions show a slight but significant decrease in fecundity (-21%) in the worms fed PFA-treated OP50 (Figure 4B). For labs interested in C. elegans aging, the effect of PFA-treated OP50 on longevity was then determined. Interestingly, the lifespan of wildtype worms fed PFA-treated OP50 was not significantly different from the worms fed mock-treated OP50 on standard nematode growth media (NGM) plates (Figure 4C), however, PFA-treated OP50 increases wildtype lifespan (+23%) when FUdR is present (Figure 4D). Feeding PFA-treated OP50 to a long-lived fmo-2 overexpressing (OE) strain²⁰ does not alter its longevity compared to wildtype worms (Figure 4E). UV-, heat-, and antibiotic-killed OP50 have also been shown to alter C. elegans lifespan. Specifically, UV-treated bacteria have been reported to extend lifespan on NGM^12,23 and on FUdR²⁴. However, there is conflicting data on the interaction of FUdR and UV-killed bacteria^25,26 that may be due to varied methods of UV-killing and validation of metabolic inactivity in the bacteria. Additionally, heat-killed bacteria extend lifespan on NGM²⁷, and antibiotics extend lifespan on NGM¹⁵ and in liquid culture²⁸.

Figure 2. Treatment of bacteria with PFA inhibits proliferation and metabolism. (A) Bacteria growth in colony forming units (CFU) of mock-treated and 0.5% PFA-treated OP50. (B) Extracellular acidification rate (ECAR) in mpH/min of mock-treated and 0.5% PFA-treated OP50. (C) Basal oxygen consumption rate (OCR) in pmol/min of mock-treated, 0.25% PFA-treated, and 0.5% PFA-treated HB101. (D) Basal oxygen consumption rate (OCR) in pmol/min of mock-treated, 0.25% PFA-treated, 0.5% PFA-treated, and 1.0% PFA-treated enterococcus faecalis (EF). All error bars shown in figures represent the standard error of the mean (SEM); a two-tailed t-test was used to derive p-values. **** denotes p-value < 0.0001. This figure has been modified from ¹⁶. Please click here to view a larger version of this figure.

Figure 3. Worms are attracted to PFA-treated OP50. Percent of worms attracted to (A) PFA-treated or (B) mock-treated OP50 bacterial lawn. (C) Pairwise sensitized assay testing worm preference for mock-treated and PFA-treated OP50. (D) Pumping rate (pumps per 30 s) of worms on PFA-treated or mock-treated OP50. A two-tailed t-test analysis was used to derive p-values for all comparisons. All error bars shown in figures represent the standard error of the mean (SEM); **** denotes p-value < 0.0001. Panels A-C have been modified from ¹⁶. Please click here to view a larger version of this figure.

Figure 4. Worms grow and develop on PFA-treated OP50. (A) Development time (h) of worms from egg-to-egg-laying adults on mock-treated and PFA-treated OP50. (B) Average progeny (number of worms) of worms fed mock-treated or PFA-treated OP50. (C, D) Percent alive of worms fed mock-treated or PFA-treated OP50 on (C) NGM and (D) FUdR lifespan plates. (E) Percent alive of wildtype and fmo-2 OE worms fed PFA-treated OP50. A two-tailed t-test analysis was used to derive p-values for development and fecundity comparisons. The log-rank test was used to derive p-values for lifespan comparisons. All error bars shown in figures represent the standard error of the mean (SEM); * denotes p-value < 0.05, *** denotes p-value <0.001, and **** denotes p-value < 0.0001. Panels A, B, and D have been modified from¹⁶. Please click here to view a larger version of this figure.

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 very effective at eliminating bacterial metabolism (Figure 2B), and PFA-treated bacteria exhibit decreased metabolic activity relative to antibiotic-treated bacteria¹⁹. Another benefit of PFA-treatment is that the bacteria maintain their cellular structure and nutritional profile. Consequently, worms can develop on PFA-treated bacteria, whereas feeding heat-killed bacteria from egg results in developmental arrest^13,16.

Critical steps and troubleshooting
While PFA-treating bacteria is a relatively straightforward protocol, it is important to validate that each batch of bacteria is replicatively and metabolically dead (steps 6 and 7) and to ensure the washing steps after PFA-treatment are done thoroughly (step 5). If colonies do grow in step 6, indicating the bacteria is not completely dead, it is possible to troubleshoot the concentration of PFA and duration of PFA-treatment in step 3. When optimizing the PFA-treatment protocol, a 0.5% concentration of PFA was ideal for successfully killing OP50 with minimal side effects. 0.25% PFA was not sufficient to metabolically kill all strains of bacteria (Figure 2C-D) and increasing the concentration to 0.5% PFA did not alter developmental time, lifespan, or food preference of worms on OP50 relative to 0.25% PFA. Also, 1 h of inoculation with PFA was the shortest time sufficient to prevent all bacterial growth. If bacteria are not completely killed by a 1 h inoculation with 0.5% PFA, the inoculation time and/or PFA concentration can be increased to troubleshoot killing efficiency. A second key step in the protocol is washing the bacteria after PFA-treatment (step 5). It is very important to complete all five washes and to completely remove the supernatant with each wash to ensure no formaldehyde remains in the bacteria. If worms are not growing well or not pumping on the PFA-treated bacteria, additional washes can be performed.

Limitations of the method
While PFA-killed bacteria are ideal for eliminating the confounding effects of bacterial metabolism (Figure 2B) from C. elegans studies, there are limitations to this method. Specifically, C. elegans have slightly slower development on PFA-treated bacteria relative to live bacteria (Figure 4A)¹⁶. PFA-treatment also extends worm lifespan if FUdR is present (Figure 4D), but not on NGM (Figure 4C) plates¹⁶. However, other methods also present these challenges; UV-killed bacteria also alter C. elegans lifespan. The majority of reports suggest UV-treated bacteria extend worm lifespan on NGM^12,23 and on FUdR²⁴, with other studies indicating UV-treatment does not affect lifespan²⁴ and that there is an interaction between UV-treatment and FUdR on C. elegans lifespan^25,26. In addition to the lifespan effects of PFA-treated bacteria, worms prefer to spend time on live bacteria relative to PFA-treated bacteria (Figure 3C)¹⁶ but have a higher pumping rate on the PFA-treated food (Figure 3D). This suggests that they may consume a similar amount of food or that PFA-treated bacteria are easier or harder to eat. Many of these effects are also observed with other methods of killing bacteria^12,13,14, suggesting that metabolically active bacteria decrease development time, shorten lifespan, and are more attractive to C. elegans.

Potential applications of PFA-treated bacteria
Overall, the ability to quickly and reliably kill large batches of bacteria has great potential for eliminating the confounding effects of bacterial metabolism in C. elegans studies. This ability is particularly useful for drug screens, supplementation experiments, toxicity assays, metabolomics studies, and examining the effects of microbial metabolism on host behavior. More information on the use of PFA-treated bacteria in each of these applications is below.

Drug, supplementation, and toxicity experiments: Because only one plate per batch of PFA-treated bacteria needs to be tested for killing success, PFA-killed bacteria can be added to liquid culture for high-throughput drug screening. PFA-treated bacteria can also be seeded onto solid plates with small molecules or nutrients incorporated into the agar media. Solid plates seeded with PFA-treated bacteria could also be useful for toxicity or stress-response assays. Unpublished data suggest that PFA-treated bacteria can help distinguish between the role of bacterial stress-response pathways and worm stress-resistance pathways in paraquat and tunicamycin resistance. Using PFA-killed bacteria in these drug supplementation and toxicity studies prevent bacteria from metabolizing the molecule of interest and will ensure that any phenotype observed results from a compound acting directly on C. elegans. Using metabolically dead bacteria for drug and supplementation experiments also minimizes variability in dosing, as live bacteria can metabolize different amounts of the compound of interest from experiment to experiment.

Metabolomics studies: C. elegans metabolomics studies can also be confounded by bacterial metabolism: observing changes in worm metabolism requires the absence of bacterial metabolic activity²⁹. Even small changes to the bacterial food source can alter the worm metabolome. For example, the metabolome of worms fed live washed bacteria differs from the metabolome of worms fed live unwashed bacteria¹⁶. Consuming PFA-treated bacteria also changes the worm metabolome relative to consuming live food¹⁶ but allows for comparisons between the effects of experimental conditions on C. elegans metabolism in isolation from bacterial metabolism.

Host-microbe behavioral studies: Finally, PFA-treated bacteria could be used to identify interactions between the bacterial metabolism and host behavior. Metabolites secreted by both pathogenic and non-pathogenic bacteria can be attractive or repellent to worms to affect their foraging and feeding behavior^10,11,30. Moreover, bacteria can produce neuromodulators that drive changes in locomotion³⁰. Additionally, growing C. elegans on different strains of bacteria results in altered brood size, development time, and physiology^31,32. Treating these bacteria with PFA can help determine whether the effects of a given bacteria on C. elegans require active bacterial metabolism.

Materials

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