Dehydration and clearing Flashcards
Process of removing intercellular & extracellular water from the tissue following fixation & prior to wax impregnation
dehydration
Characteristics of an Ideal Dehydrating Solution:
- It must dehydrate rapidly
- It should not remove stains
- Not evaporate very fast
- not toxic
- be able to dehydrate fatty tissues
- Not be a fire hazard
- It should not harden the tissue
- Best dehydrating agent - fast acting
- not expensive
- not poisonous
ethyl alcohol
- Primarily employed for blood and tissue
films and for smear preparation - toxic
methyl alcohol
- slow dehydrating agent
- recommended for tissues which do not require rapid processing
butyl alcohol
produces less shrinkage and hardening than ethyl alcohol
butyl alcohol
Factors to considered in dehydration:
- Size and nature of tissue
- type of fixative used
- temperature
- ratio
alcohol concentration:
make tissues hard brittle and
difficult to cut
above 80%
alcohol concentration:
liable to produce considerable
shrinkage and hardening of tissues leading
to distortion
85-95%
tend to harden only the surface of the tissue while the deeper parts are not completely penetrated
95% or absolute alcohol
Gen. Schedule for Alcohol Dehydration
70% alcohol - 6 hrs
95% alcohol - 12 hrs
100% alcohol - 2 hrs
100% alcohol - 1 hr
100% alcohol - 1 hr
dehydration sequence for
specimens not more than 4mm thick
70% - 15 mins
90% - 15 mins
100% - 15 mins
100% - 15 mins
100% - 30 mins
100% - 45 mins
Cheap, rapid acting dehydrating agent.
acetone
Expensive and extremely dangerous
dioxane (diethyl dioxide)
dehydrates in 30 mins to 2 hours
acetone
Used to dehydrate sections and smears
triethyl phosphate
It may be used for demixing, clearing and dehydrating paraffin sections
before and after staining
tetrahydrofuran
Clear, colorless highly flammable and extremely volatile fluid
acetone
Not recommended for routine dehydration purposes
acetone
Tissues can be left for long periods of time without affecting the
consistency or staining properties of the specimen
dioxane
The tissue may be transferred from water or normal saline directly to cellosolve and stored in it for months without producing hardening or distortion
cellosolve
combustible at 110°F to 120°F and is toxic
ethylene glycol
Triethyl Phosphate is soluble in
- water
-alcohol - ether
- benzene
- chloroform
- acetone
- xylene
removes water
triethyl phosphate
instead of ethylene glycol ether you can use
propylene based glycol
It both dehydrates and clears tissues since it is miscible in water and
paraffin
tetrahydrofuran (THF)
Additives to Dehydrating Agentsd
- 4% Phenol
- glycerol alcohol mixture or in “Molliflex)
Tissue processing for transmission electron microscopy
ethanol - dehydrating solvent
propylene oxide - transition fluid
good substitute for propylene
Acetonitrile
non-carcinogenic, less toxic and not as flammable as propylene oxide
Acetonitrile
dehydrating and clearing agent
DIOXANE
Tetrahydrofuran
The tissue may be transferred from water or normal saline directly to cellosolve and stored in it for
months without producing hardening or distortion.
Cellosolve (Ethylene Glycol Monoethyl Ether)
the transition step between
dehydration and infiltration with the
embedding medium.
clearing
Most commonly used clearing agents
xylene, dioxane, chloroform and cedarwood oil.
Recommended for routine purposes
- toluene
- xylene
Not recommended for routine purposes
- aniline oil
- cedarwood oil
- clove oil
Unsuitable for routine clearing
- clove oil
Expensive
- toluene
- cedarwood oil
- clove oil
Make tissues transparent
- xylene
- benzene
- cedarwood oil
Does not make tissue transparent
- chloroform
- carbon tetrachloride
Highly flammable
- xylene
- benzene
Does not dissolve aniline dyes
- xylene
- cedarwood oil
