Dehydration & Clearing - Histo

Question 1

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

Answer 1

Dehydration

Question 2

Alcohol increasing concentration used for dehydration

Answer 2

70% —> 95% —> 100%

Question 3

give few characteristics of an ideal dehydrating solution

Answer 3

1. Dehydrates rapidly without producing considerable shrinkage or distortion in tissues
2. should not evaporate fast
3. dehydrate even fatty tissues
4. not harden tissues excessively
5. not remove stains
6. should not be toxic to the body
7. not a fire hazard

Question 4

enumerate the commonly used dehydrating agents.

Answer 4

1. Alcohol (most common)
2. Acetone
3. Dioxane
4. Triethyl phosphate
5. Tetrahydrofuran

Question 5

it is important to keep dehydration times as ______ as possible to minimize risk of extracting cellular constituents

Answer 5

brief

Question 6

what is the general rule in tissue dehydration process?

Answer 6

Whatever dehydrating agent is used, the amount of each stage should not be less than 10 times the volume of the tissue to ensure complete penetration

Question 7

most commonly used dehydrating agent

Answer 7

Alcohol

Question 8

an alcohol recommended for routine process, fast-acting and it easily penetrates tissue, also relatively cheap

Answer 8

Ethyl alcohol (ethanol)

Question 9

a toxic dehydrating agent used for blood and tissue film smear prep

Answer 9

Methyl alcohol (methanol)

Question 10

a slow dehydrating agent, used in plants and animal micro-tech

Answer 10

Butyl alcohol (butanol)

Question 11

initially, very concentrated alcohol is used that eventually results in a relatively unequal impregnation of tissue consequently leading to poor cutting of sections

what is the remedy?

Answer 11

Start at 70% or lower concentrations of alcohol, then gradually increasing to 95%

Question 12

what will happen to the tissue if its storage is prolonged in lower concentrations?

Answer 12

Tends to macerate tissues

Question 13

what temp hastens dehydration time when using alcohol as the dehydrating agent?

Answer 13

37C

Question 14

rapid yet poorly penetrates tissue, more miscible to epoxy, removes lipids, extreme volatility and flammability, not reco as routine

Answer 14

Acetone

Question 15

excellent dehydrating and clearing agent, readily miscible with water, alcohol, xylol, and melted paraffin. extremely dangerous, expensive

Answer 15

dioxane

Question 16

dehydrates rapidly, stored for mos without producing hardening or distortion, combustible at 110-120F, toxic by inhalation (vulnerable particularly are reproductive, fetal, urinary, and blood systems)

Answer 16

Cellosolve (ethylene glycol monoethyl ether)

Question 17

If the use of cellosolve cannot be avoided, what can be used instead?

Answer 17

propylene-based glycol ethers

Question 18

tissues are fixed, washed, and transferred directly into this dehy agent, it removes water readily with little distortion, dehydrates sections and smears

Answer 18

Triethyl phosphate

Question 19

Dehydrates and clears, miscible with water and paraffin, toxic when inhaled of ingested

Answer 19

Tetrahydrofuran (THF)

Question 20

acts as softener for hard tissues e.g. nails, tendons, fibrous tissues

Answer 20

Phenol 4%

Question 21

Hard tissues can be immersed in this mixture of glycerol and alcohol

Answer 21

Molliflex

Question 22

Process whereby alcohol or a dehydrating agent is removed from tissues and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated

Answer 22

Clearing

Question 23

aka de-alcoholization

Answer 23

Clearing

Question 24

Used when tissue is to be cleared directly from water, as in frozen section

Answer 24

Glycerin and gum syrup

Question 25

Give few characteristics of a good clearing agent.

Answer 25

1. Miscible with alcohol
2. Miscible with, and easily removed by melted paraffin, and/or by mounting medium
3. not produce excessive shrinkage, hardening, or damage to tissues
4. not dissolve out aniline dyes
5. not evaporate quickly in a water bath
6. should make tissues transparent

Question 26

most common clearing agent

Answer 26

Xylene

Question 27

For urgent biopsies, clears 15-30mins, highly flammable, milky in incomp dehy tissue, cheap

Answer 27

Xylene (most common)

Question 28

Reco as routine, fairly rapid, emits fumes if highly concentrated, relatively slower than benzene and xylene, used as substitute for xylene, clears 1-2hrs, expensive

Answer 28

Toluene

Question 29

Preferred by some, urgent biopsies 15-60mins, highly flammable, carcinogenic, dmg BM results to aplastic anemia

Answer 29

Benzene

Question 30

Reco for routine work, slower than xylene but less brittle, can process up to 1cm thickness, for tough tissues, does not make tissue transparent or translucent, toxic to liver, tissues tend to float, difficult to remove from paraffin

Answer 30

Chloroform

Question 31

Extremely slow, not reco for routine, clears both paraffin and celloidin (5-6days), reco for CNS and cytological studies, 2-3 days, improves cutting of sections, milky upon prolonged use, quality not uniform, also tissue initially float

Answer 31

Cedarwood oil

Question 32

Reco for embryos, insects, very delicate spx,.. not normally utilized, able to clear 70% alcohol without excessive shrinkage or damage

Answer 32

Aniline oil

Question 33

Unsuitable for routine clearing, wax impregnation after using this clearing agent is slow and difficult,.. quality not guaranteed due to its tendency to become adulterated

Answer 33

Clove oil

Question 34

clearing agent used for tissue embedding, very similar with chloroform but relatively cheaper, highly toxic

Answer 34

Carbon tetrachloride

Question 35

Slow-acting dehydrating agents, used when double embedding is required

Answer 35

Methyl benzoate and methyl salicylate