HPCT311 Midterms Lesson 4: Dehydration and Clearing

The removal of intracellular and extracellular water from the tissue following fixation and prior to wax impregnation.

Dehydration

TRUE OR FALSE

It is important to keep the dehydration times as brief as possible to minimize the risk of extracting cellular constituents.

TRUE

Steps in Dehydration

1. Place in 70% ethanol

2. Place in 95% ethanol

3. Place in 100% ethanol

For delicate tissues, how do you start with the dehydration process?

Start with 30% ethanol

What are the characteristics of an ideal dehydrating solution?

1. It must dehydrate rapidly

2. Not evaporate very fast

3. Be able to dehydrate fatty tissues

4. It should not harden the tissue

5. It should not remove stains

6. Not toxic

7. Not be a fire hazard

What are the commonly used dehydrating agents?

1. Alcohol (Ethyl alcohol, methyl alcohol)

2. Acetone

3. Dioxane

4. Cellosolve

5. Triethyl Phosphate

6. Tetrahydrofuran

Concentration of alcohol, wherein it is liable to produce considerable shrinkage and hardening of tissues leading to distortion

85-95%

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

95% or absolute alochol

Prolonged storage of tissues in this concentration of alcohol tends to macerate the tissue

Lower concentration (below 70%)

Temperature at which alcohol hastens dehydration time of tissues.

37°C

A colorless, clear and flammable fluid Dehydrating reagent recommended for routine dehydration

Ethyl Alcohol

A dehydrating agent considered to be the best dehydrating agent because it is fast-acting.

Ethyl alcohol

A toxic dehydrating agent, recommended for blood and tissue films and for smear preparations.

Methyl Alcohol

Dehydrating agent which is utilized in plant and animal microtechniques

Butyl Alcohol

MODIFIED TRUE OR FALSE:

Statement 1: Butyl alcohol is a slow dehydrating agent, thus produceing less shrinkage and hardening.

Statement 2: It is required for tissues needed for immediate processing for results

A. Statement 1 is true

B. Statement 2 is false

C. Both statements are true

D. Both statements are false

A.

Alcoholic dehydrating agent recommended for tissues who do not require rapid processing

Butyl Alcohol

To ensure complete dehydration, a layer of _________ about 1⁄4 deep is placed in the bottom of the container and covered with filter paper.

anhydrous copper sulfate

A _________ will indicate full saturation of dehydrating fluids with water

blue discoloration of copper sulfate crystals

Arrange this process of ensuring complete dehydration from first to last.

1. A blue discoloration of copper sulfate crystals will indicate full saturation of dehydrating fluids with water

2. a layer of anhydrous copper sulfate, about 1⁄4 deep is placed in the bottom of the container and covered with filter paper.

3. Alcohol is discarded and changed with fresh solution.

4. This will accelerate dehydration by removing water from the dehydrating fluid.

A. 2,4,1,3

B. 3,2,4,1

C. 2,1,3,4

D. 2,3,1,4

D

An excellent dehydrating and clearing agent wherein tissues can be left for long periods of time without affecting the consistency or staining properties of the specimen

Dioxane (Diethylene Dioxide)

MODIFIED TRUE OR FALSE

Statement 1: Tissue sections dehydrate with dioxane tend to ribbon perfectly.

Statement 2: However, it is expensive and extremely dangerous to handle.

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

B.

A cheap dehydrating agent which is in action but penetrates tissues poorly and causes brittleness in tissues that are prolonged dehydrated.

Acetone

MODIFIED TRUE OR FALSE

Statement 1: Acetone produces less tissue shrinkage.

Statement 2: Hence, it is recommended for routine dehydration purposes.

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

D.

A dehydrating agent wherein tissues can be stored in for months without

producing hardening or distortion.

Cellosolve (Ethylene Glycol Monoethyl ether)

Temperature at which ethylene glycol ether is combustible

110°F to 120°F

A dehydrating agent which removes water very readily and produces very little distortion and hardening of tissue, which is also used to dehydrate sections and smears following certain stains and still produces minimum shrinkage

Triethyl Phosphate

A dehydrating agent which both dehydrates and clears tissues since it is miscible in water and paraffin and maye be may be used for demixing, clearing and dehydrating paraffin sections before and after staining.

Tetrahydrofuran.

MODIFIED TRUE OR FALSE:

Statement 1: Tetrahydrofuran causes less shrinkage and easier cutting of sections with fewer artifacts.

Statement 2: However, it is toxic if ingested.

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

C.

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.

Clearing

Other name for clearing

De-alcoholization

What are the most commonly used clearing agents?

1. xylene

2. toluene

3. benzene

4. chloroform

5. cedarwood oil

6. aniline oil

7. clove oil

8. carbon tetrachloride

9. methyl benzoate and methyl salicylate

The clearing agent will make microscopic tissue preparations _______ due to their _____ index of refraction.

translucent; high

What are the characteristics of a good clearing agent?

1. Miscible with alcohol to promote rapid removal for the dehydrating agent

2. Should be miscible with and easily removed by melted paraffin wax

3. Should not produce excessive shrinkage, hardening or damage of tissue.

4. Should not dissolve out aniline dyes

5. Should not evaporate quickly in a water bath

6. Should make tissues transparent

A colorless clearing agent that is most commonly used.

Xylene

It is the most rapid clearing agent, recommended for urgent biopsies.

Xylebe

MODIFIED TRUE OR FALSE:

Statement 1: Xylene cannot be used for celloidin sections because it dissolves celloidin.

Statement 2: Also, xylene is not recommended for nervous tissues and lymph nodes.

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

B.

TRUE OR FALSE

Prolonged usage of xylene makes tissues excessively hard and brittle

TRUE

A clearing agent used as a substitute to xylene or benzene

Toluene

A clearing agent recommended for routine purposes as it does not harden the tissues even if left after 24 hours

Toluene

TRUE OR FALSE

One of the main disadvantages of toluene is it tends to acidify in a partially filled vessel.

TRUE

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

Benzenw

Excessive exposure of benzene to humans can cause what condition/s?

1. Damage to bone marrow, resulting to aplastic anemia

A clearing agent which is suitable for large tissue specimens.

Chloroform

A clearing agent recommended for tough tissues, nervous tissues, lymph nodes and embryos

Chloroform

MODIFIED TRUE OR FALSE

Statement 1: Chloroform makes the tissues transparent.

Statement 2: However, it is toxic to the liver after prolonged inhalation and it is relatively slow.

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

B.

A clearing agent used to clear both paraffin and celloidin sections during embedding process

Cedarwood oil

A clearing agent recommended for central nervous system tissues and cytological studies.

Cedarwood oil

MOIDIFED TRUE OR FALSE:

Statement 1: Cedarwood oil is an extremely slow clearing agent, hence not recommended for routine purposes.

Statement 2: However, it is cheap clearing agent.

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

A.

A 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

Aniline Oil

A clearing agent which unsuitable for routing clearing purposes, wherein the quality is not guaranteed due to its tendency to become adulterated

Clove Oil

A clearing which share similar properties to chloroform but is relatively cheaper.

Carbon tetrachloride

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

Methyl Benzoate and Methyl Salicylate

TRUE OR FALSE

Carbon tetrachloride is non-toxic; hence, it is a good clearing agent.

FALSE