HISTOLOGICAL TECHNIQUE.
The methods of histological observation were at first extremely crude, consisting merely in tearing apart the tissues and examining them under the microscope. Such handling, of course, largely destroyed the relations of the different elements of the tissues to one another. The cut ting of thin sections of tissue with a razor was soon introduced, this being much facilitated by the previous hardening of the tissues in some suit able solution. Such sections were, however, diffi cult to study. owing to their transparency and the fact that the different tissue elements possess nearly the same index of refraction. The staining of sections was the next great improvement in technique. At first a single stain was used, the denser elements taking a darker shade than those of less dense structure. The discovery of what is known as differential staining, whereby the dif ferent tissue elements are stained different colors, and the introduction of an instrument for section cutting. known as the microtomc (see further below), together with improvements in the micro scope, have been the main factors in the recent rapid development of the art. At present the most commonly used technical procedures in the examination of tissues and organs is as follows: The first step is fixation. By this is meant a rapid killing of the tissue in such a way as to allow it to retain as nearly as possible the same form and relation as in the living tissue. Among the more common fixatives may he mentioned alcohol of various strengths, formalin in from 5 per cent. to 20 per cent. solutions, osmic acid, usually in 1 per cent. solution, Miiller's fluid (2.5 grains of potassium bichromate and 1 gram of sodium sulphate. dissolved in 100 cubic centi meters of water). Fleming! s fluid (a mixture of osmic, chromic, and acetic acids in water), ehrom acetic acid (1 per cent. solution in water), and corrosive sublimate in saturated aqueous solu tion. Many other fixatives are used for special purposes; thus, osmic acid is employed for the demonstration of fat and myelin. The small slices of tissue employed are allowed to remain from 12 to 4S hours in a large amount of fixative. Fixed tissues which have been fixed by any other agent than alcohol are then subjected to prolonged washing with running water. The next step is hardening. The fixing agents also act as harden ing agents if allowed to act sufficiently long. Many fixatives, however, have a detrimental ef fect upon the tissues if their action is too pro longed. It is, therefore, quite common to trans fer the tissues after proper fixation to souse new fluid for the purpose of hardening and preserva tion. The almost universal hardening and pre serving agent is alcohol. but formalin, too, in from 5 per cent. to 10 per cent. aqueous solution. is now extensively used. The first alcohol bath for plant tissues should be 3.1 per cent. or weaker, while many animal tissues will stand 50 per cent. This should be followed consecutively by 50 per cent.. 70 per cent.. 90 per cent., and 97 cent. alcohol. For long preservation SO per cent. alcohol is the most satisfactory. Next in
the process is imbedding. By this is meant the impregnation of the tissues with a liquid which afterwards hardens, thus holding the tissues in a firm mass, which can be easily cut. For this purpose paraffin and eelloidin are most commonly used. In paraffin imbedding, the tissue is lint immersed in any pure solvent of parallin, then passed to a warm solution of parallin in the solvent, and finally left in pure melted paraf fin, until thoroughly impregnated.
imbedding the tissue is transferred from alcohol to a mixture of alcohol and ether, and then placed in a solution of celloblin in a mixture of equal part- of alcohol and ether. After impregnation the paraffin is allowed to harden by cooling. or the celloidin to thicken by exposure to the air and consequent evaporation of the alcohol and ether, after which it is immersed in chloroform for hardening. The operator then proceeds to cut sections by means of the microtomr. This in strument consists essentially of a knife-carrier, which can be made to slide back and forth past a clamp to which the specimen is attached. The imbedded specimen is fastened to a block, usual ly of wood, damped in the ceurot,ute. The clamp b. so arranged that the blocked specimen can be raised any desired fraction of a milli meter, thus bringing any thickness of it above the knife. In paraffin-cutting the knife is kept dry; in celloidin section-entling it is kept flooded with alcohol. The sections are then stained for the purpose of bringing out sharply the different tissue elements. For staining the nuclei, car mine. hi•matoxylin, and various aniline dyes are commonly used. For demonstrating the other tissue elements other dyes may be used, eosin be ing much used. The procedure in staining eel loidin sections with ha.matoxylin and eosin is as follows: TIIC sections are first allowed to remain for several minutes in an aqueous solution of luematoxylin; then they are thoroughly washed with 97 per rent. alcohol, and 'dace(' for several minutes in an alcoholic solution of eosin; they are then again washed in alcohol and cleared in oil of origanum or bergamot emitaining a little eosin. From the clearing bath the specimen is lifted to a glass slide, the excess of oil is re moved by means of blotting-paper, a drop of a solution of Canada balsam is placed upon the specimen. and the whole is covered with a thin glass, called the cover-glass. By the drying and hardening of the balsam a permanent 'mount' of the specimen is secured.
The above methods of procedure are illustra tive of those applicable to general histological material. The examination of special tissues and organs requires the use of special methods of technique. This is especially true in regard to the nervous tissues, for the study of which some very elaborate 'methods have been devised, some of which will be found described in the article NF:EVOI'S SYSTEM.
For hi-tological methods as applied to plant tissues. consult Chamberlain, Methods in Mint Ifixtofomt (Chicago. 19(11): for methods in ani mal histology, consult Lee, 31 ierotornist's rade nu cum (1,onlon• 1896).