Dehydration and Clearing

Dehydration

Removal of intracellular and extracellular water from the tissue following fixation and prior to wax impregnation

10x

The amount of each stage should not be less than __ the volume of the tissue in order to ensure complete penetration of the tissue by the dehydrating agent.

1. Place in 70% ethanol

2. Place in 95% ethanol

3. Place in 100% ethanol

Steps in dehydration

30% alcohol

For delicate tissues you need to start in

85%-95% Alcohol

liable to produce considerable shrinkage and hardening of tissues leading to distortion

95% or absolute alcohol

tend to harden only the surface of the tissue while the deeper parts are not completely penetrated

lower concentration of alcohol (below 70%)

Prolonged storage in ___ tends to macerate the tissue

37°C

Temperature of ___ will hasten dehydration time

anhydrous copper sulfate

To ensure complete dehydration, a layer of ___, about ¼ 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.

blue discoloration of copper sulfate crystals

A ___ will indicate full saturation of dehydrating fluids with water

Ethyl Alcohol

● Recommended for routine dehydration

● Clear, colorless flammable fluid

● Considered to be the best dehydrating agent because it is fast-acting

Methyl Alcohol

● Toxic dehydrating agent

● Used for blood and tissue films and for smear preparations

Butyl Alcohol

● Utilized in plant and animal microtechniques

● Slow dehydrating agent

● Produces less shrinkage and hardening

● Recommended for tissues who do not require rapid processing

Dioxane (Diethylene Dioxide)

● Excellent dehydrating and clearing agent

● Produces less tissue shrinkage

● Tissues can be left for long periods of time without affecting the consistency or staining properties of the specimen

● Tissue sections dehydrated with dioxane tend to ribbon poorly

● Expensive and extremely dangerous

Acetone

● Cheap, rapid acting dehydrating agent. Dehydrates in ½ to 2 hours

● Clear, colorless highly flammable and extremely volatile fluid

● Rapid in action but penetrates tissues poorly and causes brittleness in tissues that are prolonged dehydrated

● Produces considerable tissue shrinkage

● Not recommended for routine dehydration purposes

Cellosolve (Ethylene Glycol Monoethyl Ether)

● Dehydrates rapidly

● The tissue may be transferred from water or normal saline directly to cellosolve and stored in it for months without producing hardening or distortion

● Ethylene glycol ether is combustible at 110°F to 120°F and is toxic

● Propylene based glycol ether should be used instead

Triethyl Phosphate

● It removes water very readily and produces very little distortion and hardening of tissue

● It is used to dehydrate sections and sears following certain stains and produces minimum shrinkage

Tetrahydrofuran

● It both dehydrates and clears tissues since it is miscible in water and paraffin

● 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 aniline dyes

● It is toxic if ingested or inhaled

Clearing (De-Alcoholization)

● The process whereby alcohol or a dehydrating agent is

● removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated

● Most commonly used clearing agents are xylene, dioxane, chloroform and cedarwood oil

● The clearing agent will make microscopic tissue preparations transparent due to their high index of refraction

xylene, dioxane, chloroform and cedarwood oil

Most commonly used clearing agents are

Xylene

● Colorless clearing agent that is most commonly used.

● Most rapid clearing agent, suitable for urgent biopsies

● Clearing time: ½ to 1 hour

● Makes tissues transparent

● Does not extract aniline dye

● Can be used for celloidin sections because it does not dissolve celloidin

● It is cheap

Toluene

● May be used as a substitute for xylene or benzene

● Clearing time: 1-2 hours

● Acts fairly rapidly and is recommended for routine purposes

● Tissues do not become excessively hard and brittle even if left for 24 hrs

● It is not carcinogenic

Benzene

● It is preferred as clearing agent in the embedding process of tissues because it penetrates and clears tissues rapidly

● Clearing time: 15-60 minutes

● Volatizes rapidly in paraffin oven, easily removed in the tissue

● Does not make tissues hard and brittle but it causes minimum shrinkage

● It makes tissues transparent

Chloroform

● Slower in action than xylene but causes less brittleness

● Suitable for large tissue specimens. Thicker tissue blocks (up to 1 cm) are can be processed

● Clearing time: 6-24 hours

● Recommended for tough tissues, nervous tissues, lymph nodes and embryos

● Not flammable

Cedarwood Oil

● Used to clear both paraffin and celloidin sections during embedding process

● Recommended for central nervous system tissues and cytological studies

● Clearing time: 2-3 days

● Very penetrating clearing agent

● Clears celloidin in 5-6 days

● Does not dissolve aniline dyes

● Makes tissues transparent

Aniline Oil

● Not normally utilized as a routine clearing agent

● Recommended for clearing embryos, insects and very delicate specimens due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening

Glove Oil

● Causes minimum shrinkage of tissues

● Its quality is not guaranteed due to its tendency to become adulterated ● Wax impregnation after clearing with clove oil is slow and difficult

● Tissues become brittle, aniline dyes are removed and celloidin is dissolved

● Expensive solution

● Unsuitable for routine clearing purposes

Carbon Tetrachloride

● Its properties are similar to chloroform although it is relatively cheaper

● Same disadvantage of chloroform

● It produces considerable tissue hardening and dangerous to inhale on prolonged exposure due to its highly toxic effects

Methyl Benzoate and Methyl Salicylate

● Slow-acting clearing agents that can be used when double embedding techniques are required

● Not normally utilized as a routine clearing agent

● Recommended for clearing embryos, insects and very delicate specimens due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening

Rose Oil, Carrot Oil, Olive Oil, Pine Oil

Bio alternatives for Xylene