Materials

Protocol

Infecting Snails

1. Use a 24-well tissue culture plate if the snails are 12 mm or less. Aliquot 1 mL of pond water into each well. I would go ahead and do this while the liver material is spinning.
2. Place the snails into individual wells. Make sure to mark and lids to keep track of which snail is in each well. This should be done right after you finish layering the volumetric flasks.
3. Take a 1.0 mL aliquot and put it in an 15 mL conical tube. Remove a 10 µL aliquot of the parasites and place the parasites on a slide. Put in the -20ºC for about 10 seconds. Remove from the freezer and immediately view under the microscope to get a count of parasites present. This should be repeated twice so that you get a better idea as to how many parasites are there per µL. If there are too many parasites to count, go ahead and dilute the original 1 mL aliquot with some pond water and repeat the counting process.
4. You want to add 10 parasites per well, so figure out the volume you need to add to each well, i.e. if you counted 5 parasites in 10 µL, you need 20 µL per well.
5. Add the required aliquot of parasites to individual wells that contain snails. Try to "squirt" the parasites right onto the head foot of the snail.
6. Let the snails sit with the parasites for 1.5-2 hours. However, during that time you'll need to periodically check the snails to make sure they stay within their well and in the pond water.
7. Afterwards, carefully remove the snails and put them in the appropriate cup in the snail room containing pond water and lettuce.

Note: Make sure to have a clean pair of gloves on when you return the snails to their cups. You do not want to introduce any parasites to the colonies. EtOH off your gloves after handling the infected snails and prior to entering the snail rooms.

Maintaining Schistosome-Infected Snails

Important Note: Avoid contact with parasite-contaminated water!

Note: Move snails to clean cups as needed. Replenish lettuce as needed.

1. Always have 70% EtOH available. Please make sure to wear double gloves and wear a lab coat when entering the infected snail area.
2. Label the infected snail cups with important information (snail strain, date infected, snail well and plate number, etc.).
3. Place clean cups in a plastic bin. Make sure to put as many cups as you can in the bins, even if you will not use them all. This will make the cups less susceptible to falling over. Fill the cups with pond water. Add lettuce, a small piece of styrofoam if you are collecting babies. Optional: Add a few pieces of washed coral.
4. Carefully transfer the infected snails into the clean cups with forceps. If at any time you have come in contact with the water, alcohol off your gloved hands. Make sure to place snails in the correct cups. If a snail "visited" A different cup, separate the snails and noted in the notebook. Sometimes the snail may crawl up the side of the cup and look to be dried out. Go ahead and transfer the snail into its new cup, it may survive.
5. After transferring the snails from one bin, place the forceps on paper towel and alcohol off your hands. Put the new bin on the shelf and continue with the next set of snails.
6. When you're finished transferring all the snails, alcohol off the forceps over paper towel, wipe and put the paper towel in the biohazard bucket. Alcohol off your gloved hands.
7. Place the old cups on the shelf and mark with the date.
8. Take the cups from the previous change; dump the water into the waste bucket, which contains bleach. Spritz the cups with alcohol and let it sit overnight. After 24 hours, wash with small amount of soap and rinse well.
9. Alcohol off your hands after each step. Remove your gloves in lab coat if you leaveThe infected area. Place the gloves in the biohazard bucket. Once the bucket gets full, remove an autoclave. Make sure to put in a new biohazard bag.
10. All instruments used to manipulate or hold infected snails should be soaked in alcohol.
11. Repeat the above process for the next snail change.