The overall goal of this procedure is to transform infectious CRC e into shisom. This is accomplished by first harvesting infectious CRC from the snail host. The infectious CRC is concentrated and then transformed into non-infectious shisom using a double-edged needle.
Finally, the shisom are concentrated and collected into a culture plate and incubated in RPMI at 37 degrees Celsius and 5%carbon dioxide side. Using this method transformed shisom with FU cial or cial tail contamination can be obtained. Visual demonstration of this method is critical as a transformation procedure can be difficult to learn and potentially dangerous.
Cica are infective and at a high concentration and isolation of requires delicate and precise handling to proceed safely and effectively Demonstrating their procedure will be John, a research assistant in my lab. Fill a clean 500 milliliter beaker halfway with room temperature distilled water using a standard aquarium fishnet and forceps. Transfer the infected snails into the beaker.
Place the beaker on an emergency spill tray eight to 12 inches below an incandescent light source and leave it there for one to two hours. Exposure to bright light causes the CRC to exit the snail host. To remove the snails, be sure to take extra safety precautions to avoid any contact with the infectious water, which contains CRC that can penetrate human skin directly.
Remove the snails, then replace the solution under a light source for about 15 minutes to allow any particulate waste matter to sink to the bottom of the flask. Once the waste has settled. Use a pipette to transfer the circ into a clean erlenmeyer flask, avoiding the debris at the bottom of the beaker.
Place the flask on ice at a 45 degree angle. Incubate the solution in the dark for 45 to 60 minutes to allow the CRC to settle into one corner. Once the parasites have settled, use a 50 milliliter pipette to remove most of the snat and discard it carefully.
In a beaker containing 10%bleach or 70%ethanol, gently swirl the erlenmeyer flask to re suspend the parasites. Then use a pipette to transfer the sample into sterile 50 milliliter conical tubes. Place the tubes on ice and incubate them in the dark once more for 20 minutes to allow the CRC to settle.
Once the parasites have settled, use a pipette to discard most of the supernatants, leaving five to 10 milliliters of fluid in each tube. Next, fit a syringe with a 22 gauge double-ended lure lock emulsifying needle. Slowly draw the parasite containing solution from each tube into the syringe.
Taking care not to drip the infectious mixture in the hood. Carefully turn the syringe so that the needle is pointing upward and attach a second syringe to the free end of the needle. Pass the mixture back and forth between the two syringes about 20 times to transform the crc.
Then position the syringes vertically so that the empty one is on top. Remove the top syringe carefully and disinfect it in 70%Ethanol. Deposit the parasites drop by drop into a clean, high walled Pyrex crystallization dish.
Then disinfect the needle and syringe in 70%ethanol. Next, place the dish on a white background to help visualize the parasites and add prewarm 37 degrees Celsius. Complete RPMI so that the bottom of the Pyrex dish is fully covered.
Swirl gently to collect the shisom in the center using a sterile dropper. Collect some of the concentrated shisom and transfer the parasites into a fresh 12 well cell culture plate. Repeat this, rinse step until there are no more parasites left.
Once the parasites have been transferred, fill each well up about halfway with warm RPMI to verify the presence of shisom. Visualize the plate using an inverted light microscope. At this point, most parasites should have transformed into shisom, which may be distinguished from CRC E because they're less modal and lack the characteristic cial tail.
A small number of intact CRC e severed CRC cial tails or other debris may also be present. Shta can then be grown and maintained in an incubator at 37 degrees Celsius and 5%carbon dioxide for up to six weeks with weekly media changes. Don't forget that working with infectious aria can be extremely hazardous and precautions such as double layers of glove sleeve protectors and proper use of facilities and biohazard hoods should always be taken while performing this procedure.
