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.

1. Bacteria inoculation
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Inoculate a single colony of bacteria in 100 mL of LB in a 500 mL Erlenmeyer flask. Culture the bacteria overnight in a 37 &#176;C shaker incubator.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Aliquot the bacteria in 50 mL conical tubes and store at 4 &#176;C or proceed to the next step.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Bacterial treatment with paraformaldehyde
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>In the chemical hood, add 781 &#181;L 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.</li>
	<li>Cover the flask with foil and return to the 37 &#176;C shaker incubator for 1 h. After 1 h, remove the flask from the incubator and proceed to step 5.</li>
</ol>
4. Mock-treated control
<ol>
	<li>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.</li>
	<li>Proceed to step 5.3 to complete the washing steps similar to the PFA-treated group.</li>
</ol>
5.&#160;Washing the bacteria to remove residual PFA
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Resuspend the pellet in volumes optimal for different assays. For lifespan assays, seed 60 mm plates with 200 &#181;L 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.</li>
	<li>Store the bacteria at 4 &#176;C.</li>
</ol>
6. Quality check of bacterial growth
<ol>
	<li>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.</li>
	<li>Place the plates in a 37 &#176;C incubator overnight. Check for any growth. The bacteria are considered replicatively dead when colonies do not grow on the LB plate.</li>
</ol>
7. Quality check for bacterial metabolism using a respirometer
<ol>
	<li>After the final wash and resuspension from step 5.4, confirm that the bacteria are metabolically dead using available tools such as respirometers19,20 and measuring the basal oxygen consumption rate (OCR).</li>
	<li>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.</li>
	<li>Hydrate the respirometer cartridge: Add 200 &#181;L of calibrant to all wells of a 96-well plate. Place the cartridge in the 96-well plate and incubate overnight in a 37 &#176;C incubator.</li>
	<li>Assay calibration: On the following day, place the hydrated cartridge in the machine and begin calibration.</li>
	<li>Assay test plate setup: Using a new 96-well plate, add 160 &#181;L of M9 and 40 &#181;L of the prepped bacteria (1 x 109 CFU/mL) to test wells. Add 200 &#181;L of M9 to the 4 corner wells to use as blank wells. Add 160 &#181;L of M9 and 40 &#181;L of LB used for washing and resuspending to use as negative controls. Add 200 &#181;L of M9 to the rest of the wells that will not be used.</li>
	<li>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.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65775/65775fig01.jpg" /> 
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 &#176;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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65775/65775fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Bacterial Metabolic Inactivation in Caenorhabditis elegans Using Paraformaldehyde

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The authors have nothing to disclose.

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 arrest13,16.
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 NGM12,23 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 lifespan25,26. 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 bacteria12,13,14, 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&#160;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&#160;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:&#160;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 behavior10,11,30. 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 physiology31,32. Treating these bacteria with PFA can help determine whether the effects of a given bacteria on C. elegans require active bacterial metabolism.

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&#39; 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&#39;-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&#39;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 &#62;60 &#176;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.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Method</td>
			<td>Required Materials</td>
			<td colspan="2">Scalable?</td>
			<td>Nutritional?</td>
			<td colspan="4">Effects on Worm?</td>
		</tr>
		<tr>
			<td rowspan="4">UV</td>
			<td rowspan="4">UV-crosslinker</td>
			<td colspan="2">Limited by:</td>
			<td rowspan="4">Yes</td>
			<td colspan="4">Variable effects on lifespan on NGM12, 23, 24</td>
		</tr>
		<tr>
			<td colspan="2">Number of plates that fit in UV-crosslinker</td>
			<td colspan="4">Variable effects on lifespan on FUdR24, 26, 27</td>
		</tr>
		<tr>
			<td colspan="2">Irradiation time per plate</td>
			<td colspan="4" rowspan="2">Decreased food preference16</td>
		</tr>
		<tr>
			<td colspan="2">Ability to check every plate for growth8</td>
		</tr>
		<tr>
			<td rowspan="3">Heat</td>
			<td rowspan="3">&#62;60 &#176;C incubator</td>
			<td colspan="2" rowspan="3">Yes</td>
			<td rowspan="3">No: destroys cell wall, decreased nutritional value</td>
			<td colspan="4">Developmental arrest 13</td>
		</tr>
		<tr>
			<td colspan="4">Decreased food preference13</td>
		</tr>
		<tr>
			<td colspan="4">Extends lifespan on NGM31</td>
		</tr>
		<tr>
			<td rowspan="3">Antibiotics</td>
			<td rowspan="3">Antibiotics (kanamycin, carbenicillin, etc.)</td>
			<td colspan="2" rowspan="3">Yes</td>
			<td rowspan="3">Yes</td>
			<td colspan="4">Delays growth and development15</td>
		</tr>
		<tr>
			<td colspan="4">Extends lifespan in liquid media19</td>
		</tr>
		<tr>
			<td colspan="4">Extends lifespan on NGM15</td>
		</tr>
		<tr>
			<td rowspan="3">PFA</td>
			<td rowspan="3">0.5% Paraformaldehyde</td>
			<td colspan="2" rowspan="3">Yes</td>
			<td rowspan="3">Yes</td>
			<td colspan="4">Small brood size decrease16</td>
		</tr>
		<tr>
			<td colspan="4">Small development time increase16</td>
		</tr>
		<tr>
			<td colspan="4">Decreased food preference16</td>
		</tr>
	</tbody>
</table>
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.

<table><tbody><tr><td>Aluminum Foil</td><td>Staples</td><td>2549291</td><td></td></tr><tr><td>Bunsen burner</td><td>VWR</td><td>470121-700&amp;nbsp;</td><td></td></tr><tr><td>Cell Density Meter</td><td>Denville</td><td>80-3000-45&amp;nbsp;</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorg</td><td>5430</td><td></td></tr><tr><td>Chemical fume hood</td><td>Labcono</td><td>975050411384RG</td><td></td></tr><tr><td>Conincal tubes (50 mL)</td><td>Fisher</td><td>339652</td><td></td></tr><tr><td>Cuvettes&amp;nbsp;</td><td>Fisher</td><td>14-955-127</td><td></td></tr><tr><td>E. coli OP50</td><td>CGC</td><td>OP50</td><td></td></tr><tr><td>Erlenmyer flasks</td><td>Fisher</td><td>250 mL: FB501250&lt;br/&gt; 500 mL: FB501500&lt;br/&gt; 1000 mL: FB5011000</td><td></td></tr><tr><td>Inoculation loop</td><td>Fisher</td><td>22-363-605</td><td></td></tr><tr><td>LB Agar</td><td>Fisher</td><td>BP1425500</td><td></td></tr><tr><td>Liquid waste collection bottle</td><td>Thomas Scientific</td><td>1230G50</td><td></td></tr><tr><td>Magnesium Sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>M7506</td><td></td></tr><tr><td>Paraformaldehyde (32%)</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td>Paraformaldehyde &amp;ndash; methanol free solution</td></tr><tr><td>Pipettor</td><td>Eppendorf</td><td>Eppendorf Easypet 3</td><td></td></tr><tr><td>Plastic dishes (100 mm)</td><td>Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Potassium Phosphate Monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher</td><td>P2853</td><td></td></tr><tr><td>Seahorse XF Calibrant</td><td>Agilent</td><td>100840-000</td><td></td></tr><tr><td>Seahorse XFe96 Extracellular Flux Assay Kit and Cell Culture Microplate</td><td>Agilent</td><td>101085-004</td><td></td></tr><tr><td>Serological pipettes (50 mL)</td><td>Genesee Scientific</td><td>12-107</td><td></td></tr><tr><td>Shaker incubator</td><td>Thermo</td><td>11 676 083</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher</td><td>S640-3</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Fisher</td><td>S318500</td><td></td></tr><tr><td>Sodium Phosphate Dibasic Anhydrous (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>S374-500</td><td></td></tr><tr><td>Solid waste collection bucket</td><td>M&amp;amp;M Industries</td><td>&amp;nbsp;5.0 Gallon M1 Traditional Pail</td><td></td></tr><tr><td>Tryptone</td><td>Genesee Scientific</td><td>20-251</td><td></td></tr><tr><td>Vortex</td><td>Thermo</td><td>11676331</td><td></td></tr><tr><td>Weighing balance</td><td>C Goldenwall</td><td>HZ10K6B</td><td></td></tr><tr><td>Yeast Extract</td><td>Genesee Scientific</td><td>20-255</td><td></td></tr></tbody></table>

methodology to metabolically inactivate bacteria for caenorhabditis elegans research

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.

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),&#160;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 worms13,22. 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 NGM12,23 and on FUdR24. However, there is conflicting data on the interaction of FUdR and UV-killed bacteria25,26 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.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65775/65775fig02.jpg" /> 
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 &#60; 0.0001. This figure has been modified from 16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65775/65775fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65775/65775fig03.jpg" /> 
Figure 3. Worms are&#160;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 &#60; 0.0001. Panels A-C have been modified from 16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65775/65775fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65775/65775fig04.jpg" /> 
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 &#60; 0.05, *** denotes p-value &#60;0.001, and **** denotes p-value &#60; 0.0001. Panels A, B, and D have been modified from16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65775/65775fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Advancing Caenorhabditis elegans Research Using Paraformaldehyde-Treated Bacteria at JoVE.com

Author Spotlight: Advancing Caenorhabditis elegans Research Using Paraformaldehyde-Treated Bacteria 

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.

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a ...

methodology-to-metabolically-inactivate-bacteria-for-caenorhabditis

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Biology

2.1K Views.  University of Michigan, Ann Arbor. 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.

Video: Author Spotlight: Advancing Caenorhabditis elegans Research Using Paraformaldehyde-Treated Bacteria 

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Biochemistry

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

Environment

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Developmental Biology

High-Throughput Assay to Measure Caenorhabditis elegans Food Intake and Lifespan

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

Behavior

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

Immunology and Infection

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm&#160;model.

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

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Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Automating Aggregate Quantification in Caenorhabditis elegans