Materials

Protocol

1. Turn on centrifuge so it has time to cool to 4C.
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Best to start the centrifuge ~1 hour before the start of protocol - program 2 has the correct settings saved (15 minutes, 0.5 rcf, 4C)
2. Once livers are obtained, place them in a glass petri dish and dissect any excess fat using tweezers and scalpel.
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Make note of ice packs (cold, frozen, melted, etc.), liver count and condition - they should look infected.



3. Use tweezers to transfer the livers to a blender cup, fill with enough 1.2% saline solution to just cover the livers in the blender (choose the smallest size cup - image below).

4. Blend for 20 sec. on low, 10 sec. on high, 20 sec. low, 10 sec. high (total of one minute).
5. Pour this homogenate into 50 mL conical tube(s) - fill up to 50 mL and pour any excess into the second tube. Fill the rest of the second tube with saline so that the tubes are balanced in the centrifuge.
6. Mix these by dumping both 50mL tubes and their contents into a 500 mL beaker. Swirl liquid around and refill the two 50 mL conical tubes with this mixed homogenate.
7. Centrifuge both tubes for 15 minutes, 0.5 rcf, 4C (Program 2).

8. Following centrifugation, decant the supernatant into a waste beaker and resuspend in fresh saline (fill both tubes to 50 mL and shake them vigorously to resuspend contents)
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When decanting supernatant, pour carefully - the pellet at the bottom of the tube can come loose easily - be sure not to lose any of the liver content.
9. Repeat steps 8-9 twice (3 total centrifugations with saline)
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While waiting: - Prepare the 1 L volumetric flask by covering it in aluminum foil. - Have an ice bucket prepared TIP: When the last centrifugation has 7-8 minutes left, microwave ~100 mL of artificial pond water for 20 s. Keep thermometer in the beaker and monitor the temp. It needs to be 32-34C for step 15.
10. After the third centrifugation, decant supernatant as outlined above and resuspend in artificial pond water (APW) - up to 50mL for both tubes.
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The supernatant after the third centrifugation should be clear - can be a tad cloudy, but it should be fairly clean. If not, repeat centrifugation (steps 8-9).


11. Pour contents from both 50 mL tubes into one 1 L volumetric flask (covered in aluminum foil). Use funnel for easier transfer.
12. Fill volumetric flask with room temp APW. There should be ~3 cm of the neck that is not covered in aluminum foil. (See video to visualize this).
13. Remove excess “scum” from the top of the flask (bubbles, foam, liver debris, etc.) with a disposable transfer pipette - dispose the pipette after use.
14. Use a fresh transfer pipette to add ~4 mL of warmed APW (32-34C) to the top of the flask. Create a temperature gradient by releasing warmed APW along the sides of the flask.
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4mL is an estimate, volume doesn’t matter too much. As long as you have ~3 cm of water exposed to the light (not covered by the aluminum foil).
15. Add light source to the top 3 cm of the neck, place two 15 mL conical tubes on ice and wait 5 minutes before extracting miracidia from the flask.
16. After 14 min., draw off 4 mL of miracidia from the top of the flask (they should be swimming around) and transfer to a pre-chilled 15mL conical tube using a disposable transfer pipette. Draw off another 4mL for the second tube (make sure they are both filled to 4mL). Replace this volume with warmed APW.

17. Set these tubes with the miracidia on ice and wait 15 minutes.
18. Following 15 minutes, a majority of the miracidia should have settled to the bottom of the tubes. Centrifuge these tubes for 1 minute, 0.5 rcf, 4C.
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Make sure the centrifuge is not set to program 2 anymore - deselect it and change the time to 1 min. All other settings stay the same.
19. After centrifugation, remove as much excess APW from the tube as possible using a p1000. Pipette this excess liquid into a glass petri dish to make sure there aren’t any miracidia. Repeat this for second tube.
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TIPS: (1) Hold tube up to light to make sure miracidia are congregated at the bottom - try not to pipette out miracidia. (2) If there are miracidia in the petri dish, pipette this back into the 15 mL tube and centrifuge again.
20. Fill 15 mL tube up to 1 mL with fresh APW, gently mix with p1000 and transfer all of it to a 1.5 mL tube. Repeat for the second tube.
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TIPS: (1) Have the p1000 set to 1000 µL. (2) To mix - press p1000 plunger up and down (only to the first stop) a few times and go slow. (3) Only when dispensing the liquid into the 1.5mL tube, press to the second stop.

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.