Hardening reagents for tissue preparation in histology course
The following are hardening reagents in common use for histological examination.
Alcohol as hardening reagents
As a hardening reagent this is frequently used alone. It is also used to complete the process when it has been carried to a certain stage with some other reagent. When a tissue is to be hardened in alcohol alone. It should be employed in different strengths, commencing with a weak solution (50 per cent), and passing by increases of 10 per cent. Up to methylated spirit (95 per cent) in which the tissues may be kept till they are required to be cut.
One of its chief values lies in the fact that it is so readily obtainable in the form of methylated spirit. It is suitable for the preparation of almost any tissue if used in suitable strengths. Except those in which it is required to retain the fat in situ, as in the case of the medullary sheath of nerve fibres. Here the fat-myelin-is dissolved out by alcohol.
Alcohol is found in the following different forms.
- Absolute Alcohol (containing 96 to 99 per cent)
- Rectified Spirit (84%)
- Methylated Spirit (95%)
Except in special cases, methylated spirit is employed made up with water to strengths of 50 per cent., 60 per cent., 70 per cent., or So per cent. Methylated spirit is also frequently employed as a constituent of a mixed hardening fluid.
Chromic Acid
This acid is used in strengths of from 2 to 5 per cent. It is rapid and powerful in action, with a tendency. However, to cause brittleness of the tissues. It requires a few days to work, the length of time depending on the tissue dealt with. Such as have been hardened in this fluid, and are subsequently preserved in alcohol, should be kept in the dark, as otherwise a deposit, which is difficult to remove, forms on the side of the glass. The same rule applies to all tissueswhich have been hardened in chromium salts. Before being transferred to alcohol, they should be thoroughly washed in water in order to remove the chromic acid or chromic salts
Washing in water is most satisfactorily carried out by placing the tissues in a bowl, and allowing a small stream of tap water to run into it, until the washing is completed. Several hours at least should be allowed for this, and in many cases a day or two is not too much. The process is, of course, more rapidly carried out by the above method, than by simply placing the tissues in an excess of water, and changing it from time to time; as in the former case they are subjected during the whole time to a continuous supply of fresh fluid.
Chromic Acid and Spirit
These type of reagents are sometimes em ployed for rapid hardening, in the proportion of 2 parts of the 5 per cent. solution to 1 part of spirit.
Bichromate of Potassium
Of all hardening reagents this is the most useful. It penetrates the tissues evenly, and hardens them uniformly without making them brittle, as chromic acid is liable to do. It is used in strengths of from 2 to 5 per cent. Tissues after being hardened in bichromate, as in chromic acid, should be kept in the dark if they are preserved in spirit. After having been previously thoroughly washed in water. It takes about three weeks to harden most tissues. It is especially used for the central nervous system. But in this case the time required may be doubled. Ammonium bichromate is used for the same purpose, but requires a longer time.
Miiller’s Fluid as hardening reagents
It is the form in which bichromate of potassium is most usually employed, and this fluid constitutes the hardening reagent par excellence. There is really no tissue of the body which may not be prepared with it. It has the following composition below.
- Potassium bichromate (25 grammes)
- Sodium sulphate (10 grammes)
- Water (1000 c.c.)
The hardening occupies a more or less variable time, depend ing upon the porosity of the tissue and the size of the pieces dealt with. Generally speaking, from two to five weeks are required. At any period spirit may be added tohasten or complete the process.
Muller’s Fluid and Spirit
This may be said generally to be composed of 3 parts of the former to 1. As a matter of fact they are usually combined in any proportion that may be deemed advisable in the particular case. Some times only a little spirit is added to the Muller’s fluid. Some times they are mixed in equal proportions. The more spirit the more rapid the hardening. The mixture hardens tissues more rapidly than Muller’s fluid alone.
Picric Acid
This acid is sometimes used in the form of a saturated solution. Transfer the tissues by gradations, after hardening, to methylated spirit. More frequently it is employed in the following combination.
Kleinenberg’s Solution
Saturated solution of picric acid 100cc.
Commercial sulphuric acid 2 cc.
Filter ; then add distilled water 300 c.c.
Kleinenberg’s solution is especially adapted for the prepara tion of embryonic tissues. Transfer by gradations to methylated spirit.
Chromo-acetic Acid
Chromic acid – 2 to 2·5 grammes Glacial acetic acid 1 c.c.
H2O 1000 c.c.
Chromo-acetic acid is particularly useful for the study of nuclear changes. The pieces of tissue should be small, and only subjected to the action of the reagent for a day or two. They are afterwards washed in water and transferred by successive strength stages to methylated spirit.
Osmic Acid as hardening reagents
In this acid we have an exceedingly useful, rapid hardener. It does not, however, penetrate deeply, and the pieces to be hardened should therefore be cut small. It may be used in strengths of ¼ to 1 per cent. It requires only a few hours to effect its purpose. In the case of thin membranes a still shorter time. The tissue may be washed in water and then transferred to spirit. Osmic acid stains all fatty material black. It is therefore especially used for staining adipose tissue and the myelin of the medullary sheath of nerve fibres.
Chromo-aceto-osmic Acid (Flemming’s Solution)
Chromic acid (1 per cent.) 45 c.c.
Osmic acid (2 per cent.) – 12 c.c.
Glacial acetic acid 3 c.c.
A weaker solution is sometimes used. Like chromo-acetic acid, it is especially serviceable in the study of nuclear changes. It requires from a few hours to a day or more to act. The tissues are then thoroughly washed in water, and gradually transferred to strong spirit. For the purpose of revealing the process of karyokinesis, in a specimen hardened with this reagent or with chromo-aceticacid, saffranin is the best stain.
Corrosive Sublimate
This may Le used in either a watery or alcoholic saturated solution ; the latter is the stronger. It hardens small pieces of tissue in from a half to two hours. The time required depending largely on the size of the piece of tissue. It is particularly useful for the rapid fixation of glands. When it may be advantageously employed in the form of a half saturated alcoholic solution.
- to 50 c.c. of a saturated alcoholic solution
- add 50 c.c. of methylated spirit.
The tissue should be transferred directly to alcohol to avoid precipitation of crystals of the salt.
Silver Nitrate as hardening reagents
This salt is used both for hardening and staining, and will be more especially referred to under the head ing of Staining Reagents.
The above list of hardening reagents is in no way intended to be a complete one. Only the commoner fluids are referred to, those in fact which are capable of some general application. Other methods of hardening, appropriate to special tissues, are referred to when the tissues themselves are under consideration.
With regard to the hardening of tissues generally, it is virtually impossible to lay down hard and fast rules as to the length of time required in any reagent. In practice no such rules are followed. The tissue, after being cut into pieces of an appropriate size, and washed. Then it is placed in an excess of the reagent, and its progress toward hardening is noted from day to day. Nothing but practical experience will enable the student to ascertain from the “feel”. Whether it is going on satisfactorily. A tissue ready for cutting gives, when handled, neither a sensation of softness nor hardness. But of a peculiar resilience, which becomes associated in the mind of the operator with ultimate success on the microtome.
Some of the more delicate tissues, of course, will not admit of handling, and in such cases, after the piece has been subjected to the action of the fluid for the average time. It may be supposed to be sufficiently hardened.
