Technique

Preservation and Examination of Flukes.

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.

(4) Glycerine.—Vide under methods of preservation of ova; to mount permanently, transfer to glycerine jelly; subsequently to harden the jelly, expose to formalin vapour.

Of these media, carbolic acid has the greatest refractive index excepting that of balsam. The latter may, in some cases, render structures too transparent, and it may be advisable to use only glycerine or glycerine jelly.

Preservation of Ova in Fæces, Urine, Bile, etc.

Heat some 70 per cent. alcohol in a basin to about 60 to 70° C. (until bubbles begin to appear). Add the fæces, etc., in the proportion of one part to about nine of fixative; keep stirring. Allow the sediment to settle. Transfer to a bottle with some fresh 70 per cent. alcohol.

Transference to Glycerine.—Prepare 5 per cent., 10 per cent., 20 per cent. solutions of glycerine in 70 per cent. alcohol. Pour off the alcohol in the bottle of fæces, etc., and replace by 5 per cent. glycerine solution. Allow to stand an hour or so. Then in the same way replace the 5 per cent. by a 10 per cent. glycerine, and finally by a 20 per cent. glycerine solution. When in this latter expose freely to the air (protecting from dust), so as to allow the alcohol and water to evaporate. Add a few drops of glycerine from time to time till eventually the ova are in pure glycerine. (In a very moist climate it may be necessary to use lime or calcium chloride to dry the air.) To mount permanently transfer some of the sediment to glycerine jelly.

Preservation and Examination of Cestodes.

Fixation.—(1) Saturated aqueous corrosive sublimate.—Add to this glacial acetic acid to the extent of 1 per cent. (Note this fixative will dissolve the “calcareous corpuscles”; 10 grammes of sublimate to 160 c.c. of water will give a saturated solution.) Warm the fixative to 70° to 80° C. (Avoid the use of needles.) Use plenty of fixative. Allow to act for a quarter of an hour or so. (a) Transfer to 70 per cent. alcohol. (It is advisable to remove the sublimate by the use of Lugol’s solution, or a solution containing tincture of iodine, adding this until the iodine colour is permanent.) Or (b) transfer for preservation to 10 per cent. formalin.

Or (2) 10 per cent. formalin.—In order to prevent contraction it is advisable to extend the tapeworm and keep it fixed by glass plates, or wind the worm around a wide glass tube or bottle, and then fix it.

Or (3) fix in hot alcohol.

Staining.—As under flukes. It is necessary to sacrifice portions of the tapeworm for this purpose, cutting out, e.g., mature segments, so as to study the topography of the genitalia.

Clearing.—As under flukes. To examine the hooks satisfactorily it is best to cut off the head with a sharp knife and mount. A certain amount of pressure is then advisable in order to view the hooks completely so as to measure them.

Preservation of Ova in Fæces, etc.

As under flukes.

Preservation and Examination of Nematodes.

Fixation.—(1) Thoroughly wash the worms to get rid of mucus, etc., by shaking up in warm saline (or water) till the washings are clean. Then transfer to 70 per cent. alcohol heated to about 70° C. It is absolutely necessary to use hot fixatives in order to extend the worms. If no alcohol or spirit is immediately available, drop the worms into hot water, or saline, and transfer later to 70 per cent. alcohol.

(2) Drop into hot 10 per cent. formalin.

Clearing.—(1) Carbolic acid, vide p. .

(2) Creasote, vide p. .

(3) Glycerine, vide p. .

Staining.—In case of quite small Nematodes, e.g., Anguillulidæ, carmine may be used, but as a rule staining is not advantageous.

Rolling.—In order to study the mouth parts, or bursa, etc., it is necessary to place the worm in any desired position. This is done as one would roll a penholder along the table by one’s finger placed on top of it. In the case of a worm, one edge of the cover-glass is placed over the worm, the other is supported by a strip of cardboard. By tapping the cover-glass the worm will now revolve as much as required provided it is round and straight. In certain cases it may be necessary for this purpose to cut off the head or tail. Roll these separately.

When a suitable position is got, the worm may be fixed in this position by pressure on the cover-glass, so as slightly to flatten it.

Mounting the Head.—If it is required to get an end view, it is necessary to cut off the head transversely as near the end as possible, and then mount.

Detection of Eggs (Bass and Hall).—Mix the fæces thoroughly with ten times the volume of water. Filter through gauze. Centrifugalize the filtrate. Wash the sediment and centrifugalize. Repeat twice. To sediment add CaCl2 solution, sp. gr. 1250. The eggs float to surface. Pour off surface fluid. Dilute to sp. gr. 1050. Centrifugalize. Examine sediment, which contains practically all the eggs in the stool.

Detection of Small Nematodes.—Mix the fæces thoroughly with water. Allow to settle for five minutes. Carefully decant off, or better, syphon off the fluid. Mix the sediment again with water. Allow to settle. Remove the fluid. Repeat several times. Examine the sediment in a Petri dish. As the fluid is poured off, the worms will be seen collected in the backwater. Remove them with a brush. Fix in hot 70 per cent. alcohol.

Cultivation of Larval Forms of Ancylostoma and Strongyloides.

A modification of the second method of Looss (p. ) is that of Fülleborn. A glass filter funnel is lined with linen or with cotton wool dyed black with iron-tannin. On this is placed a layer of sterile sand, and on top of this the fæces. The whole is moistened. The larvæ hatch out and wander through the meshes of the wool, appearing on the edges of the same as white threads visible to the naked eye. With a platinum needle these can be easily removed. The glass filter can be placed on a glass cylinder, and this in another large stoppered cylinder containing caustic potash solution at the bottom, so that any larvæ escaping from the funnel are killed.