General Rules

The tissue must be well hardened before being put in the decalcifying fluid, otherwise it will be much altered. The formalin method is well adapted and small pieces should be used.

An excess of fluid should be used and it should be frequently changed. After complete decalcification the tissue should be carefully washed for two or more days. It must then be rehardened before it is ready to cut. The tissue is decalcified if it allows a needle to penetrate without meeting distinct resistance.

The following are the fluids commonly used:

1. Chromic Acid And Its Salts

Miiller's fluid for small pieces of bones or embryonal bones. It is a very slow process. Can be hurried by placing in an incubator.

2. Saturated Watery Solution Of Picric Acid

Requires about three weeks for embryonal bones. Larger and older pieces take several months. Can be hastened by adding 3 to 5 per cent, of nitric acid. To remove the picric acid, wash the tissue, then place in 95 per cent, alcohol to which several drops of a saturated watery solution lithium carbonate have been added. The fluid becomes colored and more carbonate should be added until it remains completely clear.

3. Hydrochloric Acid

When used in 1 to 10 per cent, solution it works quite rapidly, but injures the tissues. Is best used as:

Ebner's fluid:

Hydrochloric acid................... ....... 2.5

Alcohol.................................... 500.0

Distilled water.............................. 100.0

Sodium chlorid............................. 2.5

This method can be hastened by increasing both the hydrochloric acid and sodium chlorid to 5 per cent.

4. Nitric Acid

Nitric Acid, in from 3 to 10 per cent, in water or formalin, is well adapted for bone tissue from adults. The alteration to the tissue is less than when corresponding solutions of hydrochloric acid are used.

Haug recommends the following on account of its more rapid and better action:

Nitric acid, c. p..........................30.0-90.0

Absolute alcohol......................... 700.0

Distilled water........................... 300.0

Sodium chlorid.......................... 2.5

5. Phloroglucin

This protects the tissues from the action of the acid, so that very strong solutions may be used. It acts very rapidly: small pieces are decalcified in one-half hour; larger ones, in several hours.

A stock solution is made consisting of:

Nitric acid, c. p.............................10 c.c.

Phloroglucin................................ 1 gm.

This is carefully dissolved by warming; is best done under a hood. To this is added 100 c.c. of a 10 per cent, aqueous solution of nitric acid.

A more slowly working mixture is:

Phloroglucin................................... 1

Nitric acid.................................... 5

Alcohol....................................... 70

Distilled water................................. 30

Thomas method is to:

1. Harden in Müller's fluid or alcohol.

2. Decalcify in:

Alcohol....................................... 5

Nitric acid.................................... 1 changing the solution very frequently.

3. Wash in alcohol.

4. Wash thoroughly in alcohol to which has been added an excess of calcium carbonate.

The decalcification requires from two to three weeks for large pieces. To remove the acid the tissue has to be in the carbonate alcohol from eight to fourteen days; should remain until there is no acid reaction with litmus-paper.

6. Trichloracetic Acid

Trichloracetic Acid, used in 5 per cent, aqueous solution and frequently changed, decalcifies in from five to seven days; generally with good results.

Injection

For the purpose of making them more easily studied the blood-vessels and other hollow structures may be filled with some injecting material that contains a stain. This procedure is not frequently used for pathologic purposes.