Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

A challenge in C elegans 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 the 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 replicatively and metabolically inactive. In addition to its consistency, PFA treated bacteria remain a palatable and nutritious food source. 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. Label 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 3000G 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 re-suspend the bacterial pellets. Repeat the centrifugation and pellet re-suspension four times. In preparation for various assays, re-suspend the pellet in suitable volumes.

For lifespan assays, seed 200 microliters of bacteria re-suspended 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, streak the Luria broth used for washing and re-suspending 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 paraformaldehyde 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, wait, 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 OP50 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 OP50 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 five fluoro two deoxyuridine. Consumption of the paraformaldehyde treated bacteria by a long-lived worm strain did not alter its longevity.