A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

The overall goal of this procedure is to infect the elegance with salmonella to examine host pathogen interactions. This is accomplished by first inhibiting the expression of a host gene by RNAi. The second step of the procedure is to infect RNA eye treated worms with salmonella.

The infection is stopped by transferring the infected worms to non-pathogenic rna eye feeding plates. The final steps are to record the survival of the infected worms. Calculate survivorship from this data, and ultimately identify genes essential for protection against salmonella.

Demonstrating the procedure will be Julie John, a technician from a laboratory. Begin by preparing the required culturing plates. First, make XLD agar plates.

They are selective for salmonella, which appear as black colonies on these plates. Be sure to resuspend XLD agar in water at one gram per milliliter before mixing it into the full volume of water. Do not autoclave.

This agar just heated with a hot plate. Proceed with making 95 millimeter diameter XLD plates, adding 25 milliliters of agar per plate. To prepare the NGM feeding plates with RNAI to make what are called RNAI plates use standard methods, briefly add ampicillin and the RNAi chemical inducer, IPTG to the media after it has been autoclaved.

Once cooled load 60 millimeter plates with 12 milliliters of agar, the plates are good for up to a month, stored at four degrees Celsius prior to their use. They're first incubated with bacteria. In this example, Beck one is the target gene as it is essential for defense against salmonella.

Start with making the RNAI and control cultures. Add a flake of frozen bacteria from the minus 80 degrees Celsius storage to two milliliters of lb with ampicillin. The bacteria should either express the RNAi or not, but still contain an empty vector.

Transfer these two cultures to an incubator and grow them up overnight. This should be done on a weekly basis so that the bacteria are always available the next day. Seed 100 microliters of the culture to the RNA eye plates.

Make at least three of each plate type experimental and control in. Incubate these plates at 37 degrees Celsius overnight. On the following day, bring the plates to room temperature.

Then transfer well fed L four wild type N two hermaphrodites to the plates. Three worms per plate will suffice. Incubate these plates at 20 degrees Celsius on the same day.

Prepare more RNAi plates with cultured bacteria. After 36 to 40 hours at 20 degrees Celsius, transfer the worms from the first RNAi plates to the new RNAi plates. Then return them to 20 degrees Celsius for another 64 hours.

Begin with streaking salmonella stock from the minus 80 degrees Celsius freezer onto an XLD plate. Incubate the plate at 37 degrees Celsius overnight. The next day, pick an isolated black colony from the plate and inoculate two milliliters of lb.

Set it to shake overnight at 37 degrees Celsius the following morning. Use the inoculated LB to seed six NGM plates. Apply 80 microliters to each plate after six hours at room temperature, the bacterial culture should have dried and will form a lawn on the NGM plates.

Then transfer the beck one RNA eye treated worms and control treated worms to the six plates. Load 40 worms onto each plate and let them incubate at 20 degrees Celsius for 48 hours. Start preparing new RNA eye plates daily at this point.

This is so they are ready in two days and every day thereafter for the survival assay. After 48 hours, transfer the infected worms back to freshly prepared RNAi plates or to empty vector control plates. Return these plates to the 20 degree Celsius incubator thereafter, every day around their egg layering time.

Score the worm survival and transfer the survivors to fresh RNA eye plates to score survival. Prob beach worm gently with a flattened platinum wire. If there is no response, the worm is dead.

During the egg laying time. Once should identify worm then died of internal hygiene. These worms should be removed in a timely fashion.

The data would be significantly ordered if this protein worm will distinctly counted as experimental worms. Once the worm stop laying eggs, it is only necessary to transfer them to fresh RNAI plates twice per week. Once the populations have died, pool their data and plot their survival on Kaplan-Meier curves.

Whatever the conclusion, perform the experiment at least twice to be sure. At 20 degrees Celsius, the median lifespan of wild type N two worms is 17 days. Salmonella infection significantly decreases the median lifespan of N two worms to 10.5 days as expected treatment with Beck one RNAi.

Decreased survivorship after salmonella infection. Mean lifespan dropped from 10.5 to nine days, and maximum lifespan was significantly shorter as well. Supportive for the role of Beck one in defending sea elegance against infection.

Animals treated with Beck one RNAi, but not treated with salmonella. Had no appreciable change in lifespan. After watching this video, you should have a good and standing of how to cover the RNA approach and the infection protocol to examine the rule of host gene in defense against semina infection.