This method can help answer key questions in the host-pathogen interaction field such as the pathogenicity and virulence of bacterial infection. The main advantage of this technique is that we can evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection. To begin wash approximately 2, 000 gravid adult worms with sterile deionized water.
Repeat the wash three times keeping the worms at the bottom of the tube. After centrifuging the worms remove the supernatant and keep the worms in 3.5 milliliters of deionized water. Add one milliliter of sodium hypochlorite and 0.5 milliliters of potassium hydroxide to the tube.
Then shake the tube for six minutes to lyse the worm bodies. After the eggs are released, add 10 milliliters of deionized water to stop the lysis. Then centrifuge the tube to pull down the eggs and remove as much of the supernatant as possible.
Wash the eggs with 15 milliliters of M9 medium at least three times. Transfer the eggs to a 3.5-centimeter dish, and incubate them at 20 degrees Celsius for one night. After this remove 10 microliters of L1 worms and M9 medium from the plate.
Count the worms and obtain the concentration of L1 worms in the M9 medium. Using a pipette take 0.5 milliliters of E.coli OP50 LB broth and spread it on an ENGM plate. Then incubate the plate at 37 degrees Celsius for 16 to 18 hours.
Cool the ENGM to room temperature. Then seed incubated L1 worms on an ENGM plate with E.coli OP50. Incubate the worms at 20 degrees Celsius until they reach the L4 stage.
First, select a single colony of each of the four Aeromonas strains and culture them according to the text protocol. Then measure the OD 600 absorbance of the bacterial broth and adjust it until the absorbance is equal to 2.0. Next spot and spread 30 microliters of bacterial broth from each strain into NGM plates.
Randomly select and transfer the previously prepared L4 worms to the NGM plates with the Aeromonas strains. Then incubate the plates at 20 degrees Celsius until the assay is complete. Every day transfer all of the living worms to a new NGM plate with bacteria.
Count the numbers of living, dead, and sensor worms every day until the last worm is dead. After culturing Aeromonas strains and measuring their absorbance as previously described, centrifuge the bacterial broth at 3, 500 g's for 15 minutes. Adjust the bacterial broth with S Medium.
And add 195 microliters of bacterial broth in the S medium to eight wells of a 96-well plate. Wash the worms off of the ENGM plate with them M9 medium. Then adjust the concentration of the worm solution to five worms per microliter.
Add five microliters of the worm solution to each of the wells of the 96-well plate. Incubate the plate on a shaker, and count the number of live worms at 24, 48 and 72 hours. After culturing Aeromonas strains and adjusting the absorbance according to the text protocol spot and spread the bacterial broth on NGM plates.
After this, transfer 50 L4 worms to each NGM plate. Every 24 hours transfer the worms to fresh NGM plates. Randomly select 10 worms and transfer them to a 2%agarose gel in M9 medium on a slide.
Finally position a coverslip on the gel and capture muscle images with a green fluorescent protein filter on a fluorescent microscope equipped with a camera. In this protocol the toxicities of four Aeromonas strains were assessed. The survival rate of C.elegans infected with Aeromonas species was highest in assays using A.caviae, and lowest in assays using A.dhakensis.
In a liquid toxicity assay survival of C.elegans decreased significantly when infected with A.dhakensis or A.hydrophilia. In the muscle necrosis assay degrees of muscle damage varied among the Aeromonas species. A.caviae caused the least muscle damage, while A.dhakensis produced the most muscle damage.
The methods described in this video offer a convenient way to differentiate virulence diversity among and within Aeromonas species. Additionally, it is also a reliable model by which to study the interaction between a pathogen and host.
