Fixation.—(Method A.) (1) Place the flukes in a test tube or small bottle a quarter full of normal saline. Shake the contents as hard as possible (the object of this is to extend the flukes) for half a minute.
(2) Add immediately an equal bulk of saturated aqueous solution of corrosive sublimate and shake again as vigorously as possible for a few minutes.
(3) Transfer when convenient to 70 per cent. alcohol. (Before staining and mounting remove the sublimate with tincture of iodine.)
(Method B.) In case of large flukes, e.g., Fasciola hepatica, Fasciolopsis buski, compress the flukes between two glass slides with rubber bands or thread. Fix in sublimate or in absolute alcohol, or in 10 per cent. formalin.
(Method C.) Place the flukes in 10 per cent. formalin solution.
Staining is successfully effected by using quite dilute solutions of carmine or hæmatein overnight. This is far preferable to using strong solutions, as it may be almost impossible to remove a too intense stain. Almost any dilute carmine solution suffices. One of the best is acetic-alum carmine (boil excess of carmine in a saturated aqueous solution of potash-alum for about fifteen minutes; add glacial acetic acid to the extent of 10 per cent.; let it stand for a week; filter). For use, dilute about thirty times with water. Place the flukes directly in the stain. Stain overnight or longer.
Differentiation.—In order to get the sharpest picture, it is best now to differentiate (but this may often be omitted) with acid alcohol (70 per cent. alcohol 100 parts, HCl 5 drops). Allow to act from one to twenty-four hours, according to the appearance of the flukes. Similarly, in staining with hæmatoxylin solution, dilute twenty to thirty times so that the water is merely tinged with the stain. Differentiate as before. After staining, dehydrate, clear, and mount in balsam if required.
Clearing and Mounting.—(1) Carbolic acid (carbolic acid 94, water 6) is a very convenient clearing agent. It may be used for stained or unstained specimens. It will clear rapidly without previous dehydration. If it is required to mount a specimen permanently, transfer from carbolic to alcohol, then cedar-wood oil (or xylol, etc.), then balsam.
(2) Creasote.—Dehydrate the specimen, stained or unstained, transfer to creasote. If it is desired to mount permanently, transfer back to alcohol, then cedar-wood oil, then balsam.
(3) Cedar-wood Oil.—Preferable to xylol or oil of cloves. Dehydrate the specimen in alcohol. To mount permanently, transfer to balsam.