In vitro excystment of Juvenile Fasciola hepatica
Paul McCusker, Rebecca Armstrong, Duncan Wells, Emily Robb, Paul McVeigh, Aaron Maule, Erin McCammick, Nathan Clarke, Erica Gardiner
Abstract
This protocol describes our excystment protocol for Fasciola hepatica supplied by Ridgeway Research Ltd. F. hepatica metacercariae (also known as mets or cysts) are supplied attached to Visking tubing with both inner and outer walls intact. This protocol builds upon methodology described by McVeigh et al. (2014) and McCusker et al. (2020) and has been tweaked by many lab members over the years. Our thanks to all who have contributed.
Steps
Outer metacercariae (met) wall removal
Pipette 300µL 50% chicken serum (CS50) onto large petri dish base and lid, spread with finger.
Fill petri dish base with 10mL RO water.
Gently lift rolled-up met sheet out of tube, place in dish base and unwrap the sheet (ensure you know which way mets are facing).
Add 100µL of RO water to petri dish lid to ensure it stays damp. Lay the met sheet onto the lid with mets facing down. NB: avoid bubble formation.
Use a scalpel to gently pop required number of mets out of outer wall (assume ~70-75% excystment rate). Outer wall is coloured brown whereas 'popped' mets are translucent. NB. only count viable mets (those with a bilobed appearance following popping).
Add 10mL water to petri dish lid before moving sheet back to petri dish base (mets facing up).
Transfer mets from petri dish lid and base to a watchglass using a serum-lined tip.
Solution Preparation
Make the following solutions and warm to 37°C:
Tube 1 – Dissolve 20mg sodium tauroglycocholate in2.5mL Excystment Salt Solution in a 15 mL falcon tube.
Tube 2 – Dissolve 20mg L-cysteine in 2.5mL 1/20 N HCl in a 15 mL falcon tube.
Bleaching mets
Swirl watchglass to gather mets in centre. Remove as much water as possible without drying out mets.
Add 1mL bleach solution to mets and start timer (time varies between sheets/strains - typically ~2 min 30 s)
While bleaching is ongoing add 1mL RO water to a new watchglass and serum line a p100 tip.
As timer approaches 0 swirl watchglass to group mets. As timer hits 0 collect mets in serum-lined p100 tip and move into watchglass with water.
Swirl mets to group, remove as much water as possible and add another 1mL RO water to wash
Repeat step 12 x5.
After final wash move mets to fresh watchglass using a serum lined p100 tip in 50µL.
Mix tubes 1 and 2 from step 8 together (some effervesce should be visible). Briefly vortex and ensure that the resulting solution is not cloudy. NB. if cloudy, do not add to worms and remake.
Pour mixed solutions onto mets and place clean watchglass on top to reduce evaporation.
Incubate in a tupperware lunchbox (line base with damp paper towel) for 1 h at 37°C.
Collection of new excysted juveniles (NEJs)
Collect NEJs with a p10 and place into pre-warmed RPMI (glass watchglass is best). After collecting excysted worms continue incubation at 37°C and collect newly excysted NEJs at 20 min intervals until 3 h after addition of excystment solutions.
