Materials

• Pen/Strep (see: recipes)
• 1.2% saline solution (18 g NaCl/1500 mL water)*
• Pond water (800 µL pond water stock/1500 mL water)*
• Blender*
• Spatula
• 15 mL conical tubes
• Ice bucket
• 1000 mL volumetric flasks
• 2000 mL beaker
• 250 mL centrifuge bottles*
• Sterile transfer pipettes
• 70% EtOH

Protocol

Extracting and hatching miracidia from mice

1. Add 1.5 mL P/S to the saline solution. Decant ~300 mL into a beaker.
2. Turn on the centrifuge so it has time to cool.
3. Place livers it in the beaker with saline and rinse well with saline to remove excess material (solution should be fairly clear/clean).
4. Drain off the saline solution and pour the livers into the appropriate blender. Add enough saline to just cover the livers in the blender. Blend for 20 seconds on low, 10 seconds on high, 20 seconds on low, and 10 seconds on high (total of one minute).
5. Divide the liver contents evenly between for 250 mL centrifuge bottles (5 livers per bottle). Rinse out the blender to remove as much liver material as possible. Add rinsed liver to the centrifuge bottles as needed. Add enough saline to each bottle so that it comes up to the 250 mL mark. Make sure to balance the bottles prior to placing them in the centrifuge. Shake the bottle as well before spinning (cover the top with paper towel).
6. Centrifuge for 15 minutes, 1600 rpm (509 g/swing bucket), 4ºC.
7. Following centrifuging, decant the saline into a large beaker. The pellet should remain at the bottom. If it breaks free from the bottom, pour off as much saline as possible without losing any of the liver sludge.
8. Add 250 mL of saline to the bottles, balance and shake well (you want to disrupt the pellet and get the liver material back into suspension).
9. Repeat step 7.
10. If you have more than 20 livers, you may need to repeat steps 8-10. The supernatant should be fairly clear.
11. You'll need one 1 L volumetric flask per 20 livers. Add approximately 200 mL of pond water to each volumetric flask. Add 1.5 mL Pen/Strep to the pond water flasks.
12. Microwave a beaker containing approximately 100 mL of pond water for 20 seconds. You want the pond water to be 32-34ºC.
13. Following the final saline wash, decant the supernatant solution, add pond water to each bottle, and shake well. Pour the contents of each centrifuge bottle to the flask.
14. Fill the flasks with pond water to the base of the neck. Remove any "scum/bubbles" from the top. You can use a large disposable pipette or a long glass pipette.
15. Gently swirl the flasks in a circular motion (to suspend the contents).
16. Fill the flask with the warm pond water using a long pasture pipette (layering). Do you want to do this slowly so that you create a "layer" Between the contents at the bottom in the neck of the flask. As you fill with the warm pond water it should become increasingly clear. Remove any debris early in the filling (make sure it's not parasites).
17. Cover the flask with tinfoil, except for a small area near the top. Place the flask next to a light source (uncovered area).
18. Place 15 mL tubes in a bucket of ice to cool.
19. Draw off the miracidia that swim to the top of the flask, only add approximately 4 mL per conical tube. After drawing off the miracidia, add back fresh, warm pond water to the volumetric flask. Put the tubes back on ice for 15 minutes.
20. Following 15 minutes, a majority of the miracidia should have settled to the bottom of the tubes. Spin the tubes for 1 minute, 4ºC, 1600 rpm. and wash with fresh pond water.

Transformation of miracidia to primary sporocysts

1. Prepare the CBSS+P/S. If it's a new bottle (100 mL), and one tube of the antibiotics (same stock concentration used for media/cell culture). Divide the contents into three 50 mL tubes, approximately 33 mL per tube (generally you will only need one tube/prep). Keep one tube in the hood, label the others and store at 4ºC until needed.
2. After spinning the tubes, immediately draw off the pond water with a pipette. Be careful not to draw up any parasites. Do this for all the tubes you have collected. Re-suspend the miracidia in approximately 1 mL of sterile CBSS+P/S (i.e. if you have four tubes, add approximately 330 µL to three tubes, make sure the parasites are in suspension, and add the contents to the tube that was not re-suspended. You'll end up with one tube in the end). Cover the tube and place back on ice for 5-6 minutes.
3. Spin the parasites again for 1 minute, 4ºC, 1600 rpm.
4. Draw off the CBSS+P/S with a pipette, once again using caution not to draw up the parasites. Re-suspend the miracidia with CBSS+P/S (approximately 5000 parasites/mL). Aliquot the parasite suspension into 24-well tissue culture plates (only use the center eight wells). Generally from 20 livers, you should end up with 14-18 wells.
5. After filling the plate, put two small pieces of tape on the side to prevent the lid from accidentally coming off, and put the in the incubator (24-26ºC).

Removal of ESP from primary sporocysts

1. After the parasites have transformed to primary sporocysts, excretory-secretory products should be removed from the plates. Transformation generally occurs between 24-48 hours after the parasites have been placed in culture.
2. Take a new tube of CBSS+P/S and let it come to room temperature.
3. Gently re-suspend the parasites by swirling the plate. Le tthe parasites settle back to the bottom, remove as much of the media as possible from each of the wells and place it into a 15 mL tube. Gently re-suspend the parasites again and combine the wells using either a fire polish glass pipette or sterile plastic pipette (combine 3-4 wells). Add fresh media to the wells which you removed the parasite suspension. After the parasites settle, once again remove as much media as possible from the well. Take care not to remove any sporocysts and add the media to the 15 mL tube.
4. Add media to the wells that contain the sporocyst, gently re-suspend, and re-distribute back into the wells which parasites were removed.
5. Place the plates containing parasites back into the incubator.
6. Spin the ESP to pellet the plates and any residual parasites. You can spin at room temperature, 2000 rpm, 6-10 minutes.
7. Remove the supernatant, place back into a 15 mL tube (make sure it is a polypropylene/"cloudy" plastic). Add the appropriate amount of protease inhibitors (Roche Mini-tab Complete Protease Inhibitors, EDTA-free). Store at -80ºC.