Dehydration, and Clearing

Question 1

Process of removing intercellular & extracellular water from the tissue following fixation & prior to wax impregnation

Answer 1

dehydration

Question 2

charac of an ideal dehydrating soln

Answer 2

• must dehydrate rapidly
• not evaporate very fast
• be able to dehydrate fatty tissues
• should not harden the tissue
• not toxic
• not be a fire hazard
• should not remove stains

Question 3

commonly used dehydrating agents

Answer 3

Alcohol
Acetone
Dioxane
Cellosolve
Triethyl Phosphate
Tetrahydrofuran

Question 4

ü for routine dehydration of tissues
ü Best dehydrating agent - fast acting
ü Not poisonous
ü Not expensive

Answer 4

Ethyl

Question 5

ü toxic
ü Primarily employed for blood and tissue films and for smear preparation

Answer 5

Methyl

Question 6

ü Utilized in Plant and Animal
Micro technique
ü Slow d.a.
ü recommend for tissues which do not require rapid processing

Answer 6

Butyl

Question 7

Factors to considered in dehydration

Answer 7

• Size and nature of tissue
  -30%
• Type of Fixative used
• Temperature
  - 370C - hasten dehydration time
• Ratio
  
  • not be less than 10X

Question 8

ü Producing less shrinkage and hardening than ethyl alcohol

Answer 8

butyl

Question 9

Effect Alcohol-Concentration

Answer 9

Ø 85%-95%
Ø above 80% -
Ø 95% or absolute alcohol
Ø Prolonged storage in lower concentration (below 70%)

Question 10

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

Answer 10

Ø 85%-95%

Question 11

• make tissues hard brittle and difficultto cut

Answer 11

above 80%

Question 12

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

Answer 12

95% or absolute alcohol

Question 13

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

Answer 13

acetone

Question 14

Prolonged storage in lower concentration tends to macerate the tissue

Answer 14

(below 70%)

Question 15

Tissue is passed through a series of progressively increasing concentrations of alcohol.

Answer 15

70% Alcohol 6 hours
95% Alcohol. 12 hours
100% Alcohol 2 hours
100% alcohol 1 hour
100% alcohol 1 hour

Question 16

A typical dehydration sequence for specimens not more than 4mm thick

Answer 16

70% ethanol 15 min
90% ethanol 15 min
100% ethanol 15 min
100% ethanol 15 min
100% ethanol 30 min
100% ethanol 45 min

Question 17

Cheap, rapid acting dehydrating agent.
Ø Dehydrates in __
Ø Clear, colorless highly flammable and extremely volatile fluid.
Ø Produces considerable tissue shrinkage
Ø Not recommended for routine dehydration purposes.

Answer 17

Acetone
1⁄2 to 2 hours

Question 18

Excellent dehydrating and clearing agent
Ø Produces less tissue shrinkage
Ø tend to ribbon poorly
Ø Expensive and extremely dangerous

Answer 18

Dioxane (Diethyl dioxide)

Question 19

Dehydrates rapidly
The tissue may be transferred from__ or __ directly to it and stored in it for
months without producing hardening or distortion.
Ø CAUTION: is combustible at __ and is toxic.
Ø __ should be used instead.

Answer 19

cellosolve ( Ethylene glycol Monoethyl ether)
water or normal saline
- 110°Fto120°F
- Propylene based glycol ether

Question 20

Removes water
Ø Produces very little distortion and hardening of tissues
Ø Soluble in __,__,__,__,__,__

Answer 20

triethyl phosphate
- Alcohol, Water, Ether, Benzene, Chloroform Acetone and Xylene

Question 21

Ø lt may be used for , _ and _ paraffin sections before and after staining.
Ø It causes less shrinkage and easier cutting of sections with fewer artefacts.
Ø It does not dissolve __
Ø It is toxic if ingested or inhaled
Ø Vapors causes ,,,_

Answer 21

tetrahydrofuran
demixing, clearing and dehydrating
- aniline dyes
nausea, dizziness, headache and anesthesia

Question 22

Additives to Dehydrating Agents

Answer 22

4% Phenol
Glycerol alcohol mixture or in “Molliflex

Question 23

is added to each 95% ethanol baths as part of dehydration process
- it acts as a softener for hard tissues

Answer 23

4% Phenol

Question 24

Ø is commonly accomplished using __ as a dehydrating solvent and __ as a transition fluid.
Ø Both solvents have some undesirable properties:
Ø __ is a good substitute for propylene
Ø non-carcinogenic, less toxic and not as flammable as propylene oxide
Ø excellent dehydrating agent

Answer 24

Tissue processing for transmission electron microscopy (TEM)
- ethanol
- propylene oxide
- Acetonitrile

Question 25

is the transition step between dehydration and infiltration with the embedding medium.

Answer 25

Clearing

Question 26

Process whereby alcohol is removed from the tissue & replaced with a substance that will dissolve the wax with which the tissue is impregnated (__) or the medium on which the tissue is to be mounted. (_)

Answer 26

De-alcoholization Paraffin Canada Balsam

Question 27

Most commonly used clearing agents are __,__,__,__ The clearing agent will make microscopic tissue preparations transparent due to their high index refraction.

Answer 27

xylene, dioxane, chloroform and cedarwood oil

Question 28

charac of good clearing agents

Answer 28

Miscible with alcohol to promote rapid removal for the dehydrating agent Should be miscible with and easily removed by melted paraffin wax. Should not produce excessive shrinkage, hardening or damage of tissue Should not dissolve out aniline dyes Should not evaporate quickly in a water bath Should make tissues transparent

Question 29

common clearing agents used

Answer 29

v Xylene (30 mins – 1 hr) v Toluene v Benzene v Chloroform v Cedarwood oil v Aniline oil v Clove oil v Carbon Tetrachloride

Question 30

Ø Colorless clearing agent that is most commonly used. Most rapid clearing agent, suitable for urgent biopsies Ø Clearing time: __ Ø Makes tissues transparent Ø Does not extract _ Ø Can be used for_ because it does not dissolve it Ø It is cheap.

Answer 30

xylene 1⁄2 to 1 hour aniline dye celloidin sections

Question 31

Ø Clearing time:__ Ø Acts fairly rapidly and is recommended for routine purposes Ø Tissues do not become excessively hard and brittle even if left for 24 hrs

Answer 31

toluene 1 – 2 hours

Question 32

Ø Clearing time: __ Ø Does not make tissues hard and brittle but it causes minimum shrinkage Ø It makes tissues transparent. Ø It is highly flammable Ø If section is let in it for a long time, considerable tissue shrinkage may be observed Ø Excessive exposure is toxic and carcinogenic to human. Ø It may damage the bone marrow resulting in _

Answer 32

Benzene 15 – 60 minutes aplastic anemia

Question 33

Ø Slower in action than xylene but causes less brittleness Ø Suitable for large tissue specimens. Thicker tissue blocks (up to 1 cm) are can be processed Ø Clearing time: __ Ø Recommended for __,__,__,__ Ø Not flammable

Answer 33

chloroform - 6 – 24 hours -tough tissues, nervous tissues, lymph nodes and embryos

Question 34

Ø Used to clear both paraffin and celloidin sections during embedding process Ø Recommended for __ and __. Ø Clearing time: __ Ø Very penetrating clearing agent Ø Clears __ in 5 – 6 days Ø Does not dissolve aniline dyes Ø Makes tissues transparent` Ø Extremely slow clearing agent, not recommended for routine purposes Ø Becomes milky upon prolonged storage and should be filtered before use Ø Very expensive

Answer 34

cedar wood oil central nervous system tissues and cytological studies 2 – 3 days celloidin

Question 35

Ø Not normally utilized as a routine clearing agent Ø Recommended for clearing_,_ and _ due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening

Answer 35

aniline oil embryos, insects and very delicate specimens

Question 36

Ø Causes minimum shrinkage of tissues Ø Its quality is not guaranteed due to its tendency to become adulterated Ø Wax impregnation after clearing with it is slow and difficult Ø Tissues become brittle, _ are removed and _ is dissolved Ø Expensive solution Ø Unsuitable for routine clearing purposes

Answer 36

clove oil aniline dyes celloidin

Question 37

Ø Its properties are similar to chloroform Ø Same disadvantage of chloroform Ø It produces considerable tissue hardening and dangerous to inhale on prolonged exposure due to its highly toxic effects

Answer 37

carbon tetrachloride

Question 38

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

Answer 38

Methyl Benzoate and Methyl Salicylate

Question 39

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

Answer 39

dioxane (diethyl dioxide)

Question 40

Used to dehydrate sections and smears

Answer 40

triethyl phosphate

Question 41

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

Answer 41

tetrahydrofuran

Question 42

Ø Highly flammable Ø If used longer than 3 hours, it will make the tissues excessively hard and brittle Ø Not suitable for _ and _ Ø becomes milky when an incompletely dehydrated tissue is immersed in it.

Answer 42

xylene nervous tissues and lymph nodes

Question 43

May be used as a substitute for xylene or benzene Ø It is not carcinogenic Ø Relatively slower than xylene and benzene Ø More expensive.

Answer 43

toluene

Question 44

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

Answer 44

benzene

Question 45

Ø Relatively toxic to the _ after prolonged inhalation Ø Wax impregnation after clearing is relatively slow Ø Does not make tissues transparent Ø Difficult to remove from paraffin sections because it is not very volatile Ø Complete clearing is difficult to evaluate

Answer 45

chloroform liver