Demonstration of Alkaline Phosphatase Activity in Various Cercaria

    Trematode miracidia and cercariae produce a variety of digestive enzymes which are used to physiologically digest a pathway into an intermediate host.  A variety of enzymes are believed to be present, including the phosphatases.  The Gomori calcium-cobalt method for detection of alkaline phosphatase is a histochemical method of localizing the site of alkaline phosphatase activity.  This stain technique involves a series of inorganic replacement reactions, the final reaction resulting in the formation of an insoluble, visible precipitate--thereby pinpointing the original site of enzyme activity. 

SOLUTIONS

    I.  3% sodium beta-glycerophosphate
    II.  2% calcium chloride
    III.  10% magnesium chloride
    IV.  2% cobalt sulfate
    V.   1 % ammonium sulfide (1ml/100 ml distilled water)
SUBSTRATE SOLUTION
    10 ml of #I, 10 drops of #III, and 1 gram of sodium barbital with the pH of the solution adjusted to 9.2 with 0.1 N NaOH.  Adjust the volume to 50 ml with distilled water

PROCEDURE

1.  Obtain cercariae from river or pond snails that have a history of containing cercariae.  If none are available, Carolina Biological Supply has cercaria-laden snails available. 
2.  Tease apart the snails and obtain the cercariae from the debris. Transfer the cercariae to depression slides filled with distilled water. Wash by pipetting the distilled water off and replacing it with fresh distilled water.  Perform this act a number of times, until the cercaria are free of snail tissue debris.
3.  Fix the cercariae with buffered formalin with 1% calcium chloride added for 10 minutes.
4.  Remove the fixative and wash with distilled water and number of times, removing the excess fixative.  Pasteur pipettes should be used in the fluid transfers.  Small bore Pasteur pipettes can be created by heating the tips of 9 inch pipettes in a Bunsen burner flame and pulling them when they are molten to create a bore small enough to handle cercariae one-at-a time. 
5.  Incubate the cercariae in the filtered, sodium beta-glycerophosphate substrate (listed above)  for 2 hours at 37 C in a depression slide.  Make sure the depression slide is covered or the substrate will totally evaporate. 
6.  Remove the substrate and wash repeatedly with distilled water. 
7.  Treat with ammonium sulfide for 2 minutes.
8.  Wash repeatedly with distilled water.
9.  Dehydrate through a graded series of ethanols to absolute ethanol.
10.  Clear in xylene for 2 minutes (xylene is carcinogenic and should only be used in a fume hood).
11.  Mount with permount.
12.  Photograph the microscopic image.

If the enzyme, alkaline phosphatase is present, it will  remove the phosphate group from the sodium glycerophosphate in the substrate used in incubating the cercariae.  The calcium ions will combine with the phosphate ions producing calcium phosphate.  Subsequent treatment with cobalt sulfate will enact a transfer reaction resulting in  the cobalt ions combining with the phosphate, creating cobalt phosphate.  With the addition of ammonium sulfide, the cobalt will claim the sulfide ion and create a black precipitate of cobalt sulfide.  That precipitate will mark the site of the original alkaline phosphatase presence.