bacterial inoculation and paraformaldehyde inactivation for caenorhabditis elegans research

Bacterial Metabolic Inactivation in &lt;em&gt;Caenorhabditis elegans&lt;/em&gt; Using Paraformaldehyde

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.

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

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

A challenge in sea elegance research is isolating the effects of a live bacterial diet from the effects of experimental manipulations. It can be difficult to determine whether the manipulation directly impacts the host or indirectly impacts the worms through a host microbiome interactions. Paraformaldehyde treated bacteria address the challenge of isolating the effects of bacterial metabolism from host physiology.Paraformaldehyde treatment is high throughput and consistently renders the bacteria replicate and metabolically inactive. In addition to its consistency PFA treated bacteria remain a palatable and nutritious food source.

Watch this Scientific Journal Video about Bacterial Inoculation and Paraformaldehyde Inactivation for  Caenorhabditis elegans  Research at JoVE.com

Bacterial Inoculation and Paraformaldehyde Inactivation for  Caenorhabditis elegans  Research  

Bacterial Inoculation and Paraformaldehyde Inactivation for Caenorhabditis elegans Research

Begin by inoculating a single bacterial colony into 200 milliliters of sterilized Luria broth in a 500-milliliter Erlenmeyer flask. Culture the bacteria overnight in a shaker incubator set at 37 degrees. After 14 hours of culture, check the optical density of the bacteria at 600 nanometers.Aliquot the bacteria into 50-milliliter conical tubes and store them at four degrees Celsius for further use. Using a serological pipette, transfer 50 milliliters of the bacterial culture into a new 250-milliliter Erlenmeyer flask Labeled the tubes for live or mock-treated control. Use a serological pipette to transfer 50 milliliters of the bacteria to a 50-milliliter conical tube for the mock-treated control before washing.In the chemical hood, add 781 microliters of paraformaldehyde to the bacterial culture to achieve a final concentration of 0.5%Dispose of the used tip in a solid waste chemical hazard container. Next cover the flask with a sheet of foil and place it in a shaker incubator at 37 degrees Celsius for one hour. Once incubation is complete, wash the bacteria to remove any residual paraformaldehyde.For the removal of the residual paraformaldehyde, using a serological pipette, transfer the treated bacteria from the Erlenmeyer flask to a 50-milliliter conical tube. Next centrifuge the treated bacteria at approximately 3000 G for 20 minutes. Discard the supernatant into a liquid waste chemical hazard container inside the chemical hood.Now add 25 milliliters of the Luria broth to the treated and control bacterial pellets, and vortex to resuspend the bacterial pellets. Repeat the centrifugation and pellet resuspension four times. In preparation for various assays, resuspend the pellet in suitable volumes.For lifespan assays. Seed 200 microliters of bacteria resuspended in 10 milliliters of Luria broth onto 60-millimeter plates. Store the bacteria at four degrees Celsius.To perform a quality check of the bacterial growth, streak a Luria broth plate with a prepared bacteria using a sterile pipette tip. To verify contamination, steak the Luria broth used for washing and resuspending on a separate plate. Next place the plates in a 37 degrees Celsius incubator overnight.The next day, check for any bacterial growth. Save the bacteria if paraform aldehyde preparation is successful and colonies fail to grow on the LB plate. To check the bacterial metabolic activity with a respirometer, first hydrate the cartridge by adding 200 microliters of calibrant to all wells on a 96-well plate.Place the cartridge in the 96-well plate and incubate overnight at 37 degrees Celsius. Set the machine to mix, weight, measure, and loop. The next day, place the hydrated cartridge into the machine.To a new 96-well plate, add 160 microliters of M9 to the test wells, followed by 40 microliters of prepped bacteria. Add 200 microliters of M9 into the four corner wells to act as blanks. Dispense 160 microliters of M9 and 40 microliters of Luria broth as negative controls.Finally, pipette 200 microliters of M9 into the remaining unused wells. Then replace the ejected calibration plate with the assay plate before running the program. The results will be displayed as the oxygen consumption rate.Different strains exhibited different inactivation response times to paraformaldehyde. For example, exposure of HB101 bacteria to 0.25%for one hour is sufficient to render it metabolically inactive. Enterococcus faecalis required a 1.0%concentration for consistent effect.Caenorhabditis elegans worms exhibited similar preferences toward the treated OP 50 strains as well as the mock-treated control. However, a sensitive pairwise assay revealed a stronger preference for the mock-treated control. Worms on treated OP 50 have a higher pumping rate than worms on the mock-treated control.Consumption of treated bacteria resulted in delayed development time in wild-type worms, as well as lowered egg laying capacity. The lifespan of the worms was not significantly different from each other, regardless of the food source. However, the paraformaldehyde-treated bacteria increased the worm lifespan in the presence of 5-Fluoro-2'deoxyuridine Consumption of the paraformaldehyde-treated bacteria by a long-lived worm strain did not alter its longevity.

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110 visualizações.  University of Michigan, Ann Arbor. Comece inoculando uma única colônia bacteriana em 200 mililitros de caldo Luria esterilizado em um frasco Erlenmeyer de 500 mililitros. Cultive as bactérias durante a noite em uma incubadora de agitadores a 37 graus. Após 14 horas de cultivo, verificar a densidade óptica das bactérias a 600 nanômetros.Aliquot as bactérias em tubos cônicos de 50 mililitros e armazená-los a quatro graus Celsius para uso posterior. Usando uma pipeta sorológica, transfira 50 mililitros da cultur...