Determination of Epidermal Plate Configuration in the Miracidium of Megalodiscus temperatus

        The miracidia of trematodes have an external surface comprised of ciliated plates (epidermal plates) which are arranged in a regular fashion.  The regularity is such that in the past, some authors chose to classify the adult trematodes relative to the epidermal plate configuration of their miracidia.  The object of this exercise is to demonstrate the epidermal plate configuration of the miracidium of Megalodiscus temperatus, the rectal fluke of frogs.

PROCEDURE
 

1.  Excise the recta of a number of Rana pipiens and place them in Syracuse dishes with distilled water. 
2.  Tease the recta apart and locate the adult flukes.  Megalodiscus temperatus is characterized by two red-brown caeca and a large terminal acetabulum.
3.  Transfer the adult flukes to separate dishes containing filtered aquarium water or pond water.  The adults will begin depositing numerous egg capsules.
4.  Transfer the egg capsules to a separate dish.  The miracidia will hatch within the next minutes to hours.
5.  After the greatest number of miracidia have hatched, quickly pipette hot (60-70C) freshly-prepared,  0.5% silver nitrate into the depression slide and allow the miracidia to settle to the bottom.  Circular agitation of the dish can be used to concentrate them toward the middle. 
6. Pipette (Pasteur pipettes) off the excess silver nitrate and replace it with distilled water.  Repeat this procedure several times.  This should be done while observing with a dissecting microscope to insure that the miracidia are not also pipetted off and discarded.  To reduce the possibility of inadvertently discarding miracidia, reduce the bore of the Pasteur pipette.  To do this, place the tip of a 9 inch Pasteur pipette in the flame of a Bunsen burner. When the tip is molten, pull the glass apart and the bore of the tip will be reduced.  With practice, pipettes can be modified so that individual miracidia can be pipetted "single file". 
7.  Expose the miracidia to direct sunlight for 3-10 minutes dependent upon the cloud cover that day.
8.  Dehydrate through an ascending series of ethanols, clear with xylene and mount with permount.  (Xylene is carcinogenic and should only be worked with in a fume hood).  An alternate technique to the use of permount would be to place the miracidia in a 10 % glycerine solution and allow the water to evaporate, bringing the medium gradually to pure glycerine.  The latter technique utilizes glycerine jelly as the mounting medium and a double mount technique should be employed.
9.  Photograph the miracidia shortly after they have been mounted.  The reduced silver nitrate clings to the edges of the epidermal plates, nicely delineating them.  However, as the time passes, the silver continues to be reduced, blackening the miracidia.

miracidium of Megalodiscus temperatus epidermal plates
miracidium of Megalodiscus temperatus Nile blue sulfate