When frozen, gum forms a firm non-crystalline mass, which supports the tissue on all sides. It must not be frozen too deeply, or it becomes hard and rather brittle and is apt to injure the razor. If this have occurred the surface can be softened sufficiently by breathing gently on it.
After cutting in gum, the sections are gently removed from the knife into distilled water by a soft camel’s hair brush, and left there for an hour or two, until the medium is entirely dissolved out. They may then be stained and mounted, or they may be put away in spirit for an indefinite time, and then stained and mounted.
Celloidin is for many purposes almost an ideal embedding medium. (1) It has great penetrating power; (2) it can be made of an admirable consistence for cutting purposes; (3) after sections are made it allows them to be very freely manipulated without fear of injuring them: (4) and being perfectly transparent and homogeneous in thin sections, it does not require to be removed from a section before mounting. It is insoluble in water, and in weak spirit; slightly soluble in alcohol of more than 90 per cent. strength, and very readily soluble in ether, or in a mixture of alcohol and ether. The last solvent is the one commonly employed.
The embedding solution is made thus:—
Take some pure celloidin (“Schering’s,” sold in boxes containing an ounce of shavings, is very good) and pour on it about eight times its volume of a mixture of equal parts of absolute alcohol and ether. Allow this to stand all night until the celloidin is dissolved. The solution should be made about the consistence of ordinary mucilage.
It is also convenient to have a thinner solution made by using double the proportion of alcohol and ether. Both solutions should be kept in wide mouthed stoppered bottles, and the stopper should be well greased with vaseline as an additional obstacle to the evaporation of the volatile ether.
Before embedding a specimen it is necessary to dehydrate it thoroughly for twelve to twenty-four hours in absolute alcohol. It should then be placed in a mixture containing alcohol and ether for an hour or two, and afterwards transferred to the thin solution of celloidin for twenty-four hours, and then to the thick solution for the same period. The celloidin penetrates slowly and in the case of nerve tissues and other delicate structures it is wise to give the full allowance of time for the different steps. When the tissue has been thoroughly permeated by the celloidin, it is gently removed from the celloidin and placed in position on a piece of cork of suitable size for clamping in the holder of the microtome. Celloidin is painted round the object so that it is supported on every side. It is then left exposed to the air until the surface has become firm, when the cork is placed, with the tissue downwards, in methylated spirit. The cork floats but the tissue and celloidin remain submerged. At the end of twenty-four hours the celloidin will have become semi-opaque and opalescent, and of the same consistence as hard boiled white of egg. When it is impossible to wait so long, rapid hardening of the celloidin may be secured by immersing it in methylated chloroform in place of spirit, but the slower method gives more uniformly satisfactory results.
Pieces of tissue embedded in celloidin may also be cut on a freezing microtome. After the celloidin has become firm by immersion in methylated spirit, the tissue with the celloidin round it may be cut off the cork, washed in water to remove the alcohol, and then soaked for an hour or two in gum, placed on the plate of an ether spray microtome, frozen and cut in the usual way.
Subsequent staining operations are conducted in the same way as for sections cut by hand or in gum. As celloidin is only slightly stained by hæmatoxylin, alum carmine, borax carmine, &c., it is not necessary to remove it from the sections, but it exhibits so intense a staining reaction with aniline dyes that it is necessary to remove it by treatment with alcohol and ether either before or after the staining operation.