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water for some hours, the water should be changed several times, then place it in dilute spirit made thus:Take

Methylated Spirit 2 parts, and
Water I part.

About half the quantity of this spirit mixture may be used, but the material must be well covered.

Let the material remain in this for from 24 to 36 hours, never longer than three days, and then replace it by pure methylated spirit, enough to cover the material; it may remain in this for an indefinite time, but it will often be found that the spirit becomes cloudy and full of deposits in a few days; in this case it is only necessary to change the spirit until it remains clear.

A large quantity of per cent. solution of chromic acid should be kept on hand, and it should be mixed with the spirit as required, it will be found the most useful of all the hardening agents, if it is changed at the proper time.

In some cases which will be mentioned further on, a per cent. solution of chromic acid may be used without the spirit with advantage. In other cases it may be necessary to use a solution much weaker, as a per cent. These are, however, not required for the ordinary purpose of the Histologist, but when special investigation is being made of some particular organ, it is better to try the effect of different strengths of the hardening agent to see which gives the best result in that particular case.

2. Müller's Fluid is a good hardening mixture, but requires a much longer time, taking weeks to do

what the chromic acid mixture will do in days. It is

made thus:

Take

Potass. Bichrom. 2 parts.

Soda Sulphat. I part.

Water 100 parts.

In making this solution, the ingredients should be pounded up in a mortar, and then warm water added until they are dissolved. A large quantity of the fluid should be used, about ten times the bulk of the tissue.

The advantage of this mixture is that larger pieces can be hardened in it, and it does not require changing after the first week or two, but it will take from 5 to 7 weeks to harden anything, according to its size. When it becomes cloudy it requires changing. The material, when sufficiently hardened, should be well washed and then placed in dilute spirit in the same manner as recommended after hardening in the chromic acid mixture.

3. Dilute Spirits. Many tissues can be hardened in spirit alone if they are placed in dilute spirit at first, so that the elements of which they are composed are not shrunk. This process is also used after hardening by any of the others.

Dilute spirit is made by adding I part of water to 2 parts of methylated spirit.

The material to be hardened should not be left in this mixture more than from 24 to 48 hours.

It is then transferred to pure methylated spirit, and after remaining in it for from 3 to 5 days, it is ready for cutting.

4. Bichromate of Potash. Make a 2 per cent. solution and keep it on hand, as it is very useful for many tissues that require slow hardening. A solution can be made much more quickly with warm water than cold. This solution is also very useful to place portions of morbid material in, on their removal from the body in the post-mortem room, they can afterwards be transferred to the chromic acid mixture for more rapid hardening. This solution takes from three to seven weeks to harden, according to the size of the specimen, and the frequency with which the solution is changed.

5. Bichromate of Ammonia. A 2 per cent. solution is used in precisely the same manner as the former, and is applicable to the same tissues.

6. Chromate of Ammonia. Make a 5 per cent. solution, that is I oz. of the salt to 20 oz. of water, or 5 grammes to 100 c.c., and filter.

Keep it in a stoppered bottle. When this hardening agent is used for fresh tissue, such as mesentery, a small quantity is placed in a glass vessel and the tissue immersed in it for 24 hours, it is then washed until no more colour comes away and mounted in glycerine.

For other tissues it is necessary that the material should be cut into small pieces and left in the solution for 24 to 48 hours. It is then placed in distilled water, which must be changed several times until it is no longer tinged. The hardening is completed by the spirit process (page 18).

7. Chloride of Gold.

Half per cent. solution. This is sold in small glass tubes, each containing 15 grains

of the chloride, equal to 7 grains of pure gold. Take one of these tubes and file a ring round it above the bulb, it can then be easily divided into two parts, empty the gold chloride into a 6-ounce bottle, and wash out any particles that remain with distilled water, fill up the bottle. This will be under a half per cent. solution, but answers very well. Place a small quantity of this solution in a watch glass and immerse the tissue, which must be perfectly fresh, in it, let it remain in the dark for from half to one hour or more, then place in distilled water, which must be changed several times, and exposed to diffuse daylight until it becomes a violet brown; about 24 hours will do in summer.

The tissue can then be mounted in glycerine, if it is a small thin substance, such as a tadpole's tail.

If, however, larger portions of any tissue are stained with gold chloride, the hardening will not be sufficient, and they must be further hardened by the spirit process (page 18).

Mouse-tail, stained and hardened by the gold process, may be decalcified by placing it in a half per cent. solution of chromic acid, or a saturated solution of picric acid for a few days.

The above process does very well for such small things as the tail of tadpole or mouse; but where portions of tissue are used certain modifications are required.

It is very difficult to get the gold to penetrate in the first place, and even more difficult to reduce it when this is done. For this purpose many processes have been tried. One of the best seems to be that adopted by Professor Ranvier.

He places the perfectly fresh tissue in lemon juice for five minutes before going into the gold solution. It may also be placed in lemon juice again after the gold, or in dilute acetic acid. But the best results so far, have been obtained by placing the tissue in a solution of tartaric acid until it is saturated, and then reducing by heat. Having used the lemon juice in the first place, the best plan to reduce the gold is to heat some water in a water-bath to a temperature of about 100°F. = 38° to 40°C., place the bottle containing the material in the tartaric acid solution, when it has become saturated, in the water-bath without a stopper, and let the two fluids stand at the same level. Keep at this temperature for ten minutes, and then allow it to cool gradually, wash well, and either cut sections at once or harden the tissue and cut in the usual manner.

The sections in either case must be exposed to daylight for two or three days in distilled water, which must be frequently changed.

Gold is a most provoking substance to work with, as it is not possible, seemingly, to depend on the result in any case, a brilliant specimen sometimes being made, which the same method will utterly fail to reproduce, although used in a precisely similiar

manner.

For another gold process, see Cornea. Gold chloride has a staining as well as a hardening action.

8. Silver Nitrate. Nitrate of silver possesses the property of forming a compound with the intercellu

* The tartaric acid solution must be just under saturation.

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