Exposure of snails to miracidia

 

Author Fred Lewis

Introduction

Schistosome eggs usually hatch readily when placed in fresh water, depending upon the tissues from which they are obtained. Concentrated egg pellets, when diluted in copious amounts of fresh water, can yield large numbers of miracidia for experimental infection of snails.

Equipment

Waring blender

Centrifuge

Dissecting microscope

Darkened side-arm flask (with side arm not darkened)

Hand-held counter

 

Materials and reagents

50 ml conical centrifuge tubes

Pasteur pipettes

Fine-tipped (drawn) pasteur pipettes

Aged tap water

 

Eggs of S. mansoni, S. haematobium, and S. japonicum hatch readily if placed in aged tap water. Allowing them to hatch in a petri dish works well, but it may be difficult to clean up the preparation well enough to obtain the miracidia easily. If one has a side-arm flask, the following procedure works well in obtaining a miracidial suspension relatively free from tissue debris.

Procedure

·         Mince tissue containing eggs for 30 seconds in 0.85% NaCl, using a low speed setting on a Waring blender.

·         Centrifuge homogenate for 5 minutes at 100 x g.

·         Pour off the supernate and resuspend the pellet in aged tap water that has been pre-warmed to 26ºC.

·         Place the egg suspension in a 1-liter darkened side arm flask in which the side arm is not darkened (photo-flask), and fill the flask with aged tap water.

·         Shine a light on the exposed side arm, taking care not to overheat the side arm. Since the miracidia  are  phototropic, they will begin to collect in the water of the side arm in 20-30 minutes, at which point they can be removed by a pasteur pipette.

·         Withdraw a pipette full of miracidial suspension, and place it in a petri dish with additional aged tap water.

·         Add aged tap water back into the side-arm flask to keep the volume constant.

·         With a drawn pasteur pipette and using a dissecting microscope, withdraw the appropriate number of miracidia and place with the snails in a small volume of water.

·         Incubate snails with miracidia for at least 2 hours to ensure miracidial penetration.

Follow-up comments/recommendations

Large numbers of miracidia can be obtained from the livers of mice infected for 7 weeks with 150 S. mansoni cercariae per mouse, or 20-30 S. japonicum per mouse. Miracidia can be obtained from eggs from feces of infected mammals, but they usually do not hatch as quickly in water as do those from tissues (liver and intestines).Hamsters infected for 3 ½ - 4 months with S. haematobium will have most of the recoverable eggs in the intestinal walls, rather than in the liver.

 

 Using a darkened side arm flask assures a cleaner miracidial preparation than one from which miracidia are not selectively attracted to light.

 

 

References

McMullen, D. B. and Beaver, P. 1945. Studies on schistosome dermatitis. IX. The life cycles of three dermatitis-producing schistosomes from birds and a discussion of the subfamily Bilharziellinae (Trematoda: Schistosomatidae). American Journal of Hygiene 42: 128-154.

 

Lewis, F.A., Stirewalt, M.A., Souza, C.P., and Gazzinelli, G. 1986. Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations. Journal of Parasitology 72: 813-829.