Dehydration

As soon as tissues have been fixed, and the bones and teeth have been decalcified, it is necessary to remove the water from the tissue in preparation for impregnation. This process of removing intercellular  and extracellular water from the tissue following fixation and prior to wax impregnation is known as dehydration; and the solutions utilized to make this possible are called dehydrating agents.

Characteristics of an ideal dehydrating solution

1. It must dehydrate rapidly without producing considerable shrinkage or distortion of tissues.

2. It should not evaporate very fast.

3. It should not be able to dehydrate even fatty tissues.

4. It should not harden tissues excessively

5. It should not remove stains.

6. It should not be toxic to the body.

7. It should not be a fire hazard.

Commonly used dehydrating agents

1. Alcohol

The tissue is passed thru a series of progressively increasing concentration of alcohol. Under no circumstances should a formalin–fixed tissue be transferred directly to higher grades of alcohol, e.g. 85 – 95% Alcohol because this is liable to produce considerable shrinkage and hardening of tissues leading to distortion. Concentrated alcohol (95% or absolute) tend to harden only the surface of the tissue while the deeper parts are not completely penetrated. This will result in a relatively unequal impregnation of tissue with consequently poor cutting of sections. To avoid this, 70% or lower concentrations of Alcohol, gradually increased to 95% is used.

The strength of initial Alcohol required in each concentration will depend upon the size, and nature of each tissue and fixative used. Generally, smaller and more delicate tissues require lower concentrations and shorter intervals between changes of succeeding ascending grades of alcohol. A very concentrated solution (above 80%) makes tissues hard, brittle and difficult to cut. Prolonged storage in lower concentrations of Alcohol (below 70%) tends to macerate the tissue. The tissue may be stored in 70 – 80% alcohol, although not for very long periods of time, since this may later interfere with the staining properties of the specimen.

A temperature of 37^oC will hasten dehydration time, and is especially used for tissue sections that require urgent examinations such as fragmentary biopsies. To ensure complete dehydration, a layer of Anhydrous Copper Sulfate, about ¼ inch deep is placed in the bottom of the container and covered with filter paper. This will accelerate dehydration by removing water from the dehydrating fluid. A blue discoloration of Copper Sulfate Crystals will indicate full saturation of dehydrating fluids with water. Alcohol is then discarded and changed with a fresh solution.

2. Acetone

It is a cheap, rapid–acting dehydrating agent utilized for most urgent biopsies which it dehydrates in ½ to 2 hours. Its use has been limited, however, only to small pieces of tissues due to its extreme volatility and inflammability. Because of considerable tissue shrinkage produced, acetone has not been recommended for routine purposes.

3. Dioxane (Diethyl Dioxide)

Dioxane is an excellent dehydrating agent and clearing agent readily miscible in water, melted paraffin, Alcohol and Xylol. It produces less tissue shrinkage as compared to Alcohol dehydration. Tissues can be left in this reagent for long periods of time without affecting the consistency or staining properties of the specimen. Because Dioxane is miscible with both water and paraffin, tissues may be placed directly into the solution after washing out. However, tissue sections dehydrated with dioxane tend to ribbon poorly. Aside from being expensive, dioxane is also dangerous, and this is its main disadvantage. Its vapor being expensive, dioxane is also dangerous, and this is its main disadvantage. Its vapor produces cumulative and highly toxic action in man; hence, it should be used routinely. The laboratory room should be properly ventilated, and all residues should be washed down in the sink.

The following is an example of a time for dehydration with Dioxane (Graupner’s method)

1st	Pure Dioxane Solution	1 hour
2nd	Pure Dioxane Solution	1 hour
3rd	Pure Dioxane Solution	2 hours
1st	Paraffin Wax	15 minutes
2nd	Paraffin Wax	45 minutes
3rd	Paraffin Wax	2 hours

In another method (Weiseberger’s method), the tissue is wrapped in a gauze bag and suspended in a bottle containing dioxane and a little anhydrous calcium oxide. Water is displaced from the tissue by dioxane and in turn absorbed by calcium oxide or quicklime. Dehydration period ranges from 3 – 24 hours.

Tissues which have been treated with chromate fixative, e.g., Regaud’s or Moller’s fluid, should be thoroughly washed in running tap water prior to treatment with dioxane in order to remove the chromate.

4. Cellosolve (Ethylene Glycol Monoethyl Ether)

Cellosolve dehydrates rapidly and is not harmful to the tissues. The tissue may be transferred from water to normal saline directly to cellosolve and stored in it for months without producing hardening or distortion.

5. Triethyl Phosphate

Tissues are fixed, washed and transferred directly into Triethyl Phosphate solution for dehydration. It removes water very readily and produces very little distortion and hardening of tissues. It is soluble in Alcohol, Water, Ether, Benzene, Chloroform, Acetone and Xylene.  It is used to dehydrate sections and smears following certain stains and produces minimum shrinkage.

6. Tetrahydrofuran (THF)

Tetrahydrofuran is a reagent that both dehydrates and clears tissues since it is miscible in both water and paraffin. It can dissolve many substances including fats and is in itself miscible with lower Alcohols, Ether, Chloroform, Acetone, Benzene and Xylene. It may be used for demixing, clearing and dehydrating paraffin sections before and after staining. It causes less shrinkage and easier cutting of sections with fewer artifacts. It does not dissolve out aniline dyes. In fact, most staining procedures give improved results with tetrahydrofuran. It is also non–toxic although prolonged exposure (up to 6 months) may cause conjunctival irritation. Because of this and it's rather offensive odor, processing with THF should be done in a well–ventilated room.

As a general rule, whatever dehydrating agent is used, the amount in each stage should not be less than 10 times the volume of the tissue in order to ensure complete penetration of the tissue by the dehydrating solution.