Dehydration & Clearing - Histo Flashcards
process of removing intracellular and extracellular water from the tissue following fixation and prior to wax impregnation
Dehydration
Alcohol increasing concentration used for dehydration
70% —> 95% —> 100%
give few characteristics of an ideal dehydrating solution
- Dehydrates rapidly without producing considerable shrinkage or distortion in tissues
- should not evaporate fast
- dehydrate even fatty tissues
- not harden tissues excessively
- not remove stains
- should not be toxic to the body
- not a fire hazard
enumerate the commonly used dehydrating agents.
- Alcohol (most common)
- Acetone
- Dioxane
- Triethyl phosphate
- Tetrahydrofuran
it is important to keep dehydration times as ______ as possible to minimize risk of extracting cellular constituents
brief
what is the general rule in tissue dehydration process?
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
most commonly used dehydrating agent
Alcohol
an alcohol recommended for routine process, fast-acting and it easily penetrates tissue, also relatively cheap
Ethyl alcohol (ethanol)
a toxic dehydrating agent used for blood and tissue film smear prep
Methyl alcohol (methanol)
a slow dehydrating agent, used in plants and animal micro-tech
Butyl alcohol (butanol)
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?
Start at 70% or lower concentrations of alcohol, then gradually increasing to 95%
what will happen to the tissue if its storage is prolonged in lower concentrations?
Tends to macerate tissues
what temp hastens dehydration time when using alcohol as the dehydrating agent?
37C
rapid yet poorly penetrates tissue, more miscible to epoxy, removes lipids, extreme volatility and flammability, not reco as routine
Acetone
excellent dehydrating and clearing agent, readily miscible with water, alcohol, xylol, and melted paraffin. extremely dangerous, expensive
dioxane
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)
Cellosolve (ethylene glycol monoethyl ether)
If the use of cellosolve cannot be avoided, what can be used instead?
propylene-based glycol ethers
tissues are fixed, washed, and transferred directly into this dehy agent, it removes water readily with little distortion, dehydrates sections and smears
Triethyl phosphate
Dehydrates and clears, miscible with water and paraffin, toxic when inhaled of ingested
Tetrahydrofuran (THF)
acts as softener for hard tissues e.g. nails, tendons, fibrous tissues
Phenol 4%
Hard tissues can be immersed in this mixture of glycerol and alcohol
Molliflex
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
Clearing
aka de-alcoholization
Clearing
Used when tissue is to be cleared directly from water, as in frozen section
Glycerin and gum syrup
Give few characteristics of a good clearing agent.
- Miscible with alcohol
- Miscible with, and easily removed by melted paraffin, and/or by mounting medium
- not produce excessive shrinkage, hardening, or damage to tissues
- not dissolve out aniline dyes
- not evaporate quickly in a water bath
- should make tissues transparent
most common clearing agent
Xylene
For urgent biopsies, clears 15-30mins, highly flammable, milky in incomp dehy tissue, cheap
Xylene (most common)
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
Toluene
Preferred by some, urgent biopsies 15-60mins, highly flammable, carcinogenic, dmg BM results to aplastic anemia
Benzene
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
Chloroform
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
Cedarwood oil
Reco for embryos, insects, very delicate spx,.. not normally utilized, able to clear 70% alcohol without excessive shrinkage or damage
Aniline oil
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
Clove oil
clearing agent used for tissue embedding, very similar with chloroform but relatively cheaper, highly toxic
Carbon tetrachloride
Slow-acting dehydrating agents, used when double embedding is required
Methyl benzoate and methyl salicylate