Dehydration and clearing Flashcards
Both dehydrates and clears tissues since it is miscible in both water and paraffin
Advantage
-Causes less shrinkage and easier cutting of sections with fewer artifacts. It does not dissolve out aniline dyes. In fact, most staining procedures give improved results with tetrahydrofuran.
-Does not need for clearing. It acts as both dehydrating and clearing agent.
Disadvantage
- Strong irritant; may cause conjunctival irritation (exposure up to 6 months)
- Strong odor; must be used in a well-ventilated room.
-Detrimental in the environment
Diluted by ethanol
Tetrahydrofuran
Removal of intracellular and extracellular water from the tissue following fixation and prior to wax impregnation.
Dehydration
Ratio of dehydrant to tissue
1:10
<10 tissue to dehydrant ratio leads to
Poor impregnation/infiltration
> 10 tissue to dehydrant (excessive dehydrant) ratio leads to
cell shrinkage and tissue structure distortion
Very fast evaporation of dehydrating solution causes
Iron deposition in the cell leading to false negative for hemosiderosis and removes organelles inside the cell
Dehydrating agents: “Drinking alcohol”
Advantages:
-Non- toxic
-Miscible with water
-No shrinkage when graded treatment
-Ideal for delicate tissues
Disadvantages:
-It may cause hardening of tissues when left for longer period in absolute alcohol
-Highly inflammable
Ethyl Alcohol (Ethanol)
Dehydrating agents: AKA Rubbing alcohol
Advantages:
-Excellent substitute for ethanol
-Miscible with water and organic solvents
Disadvantages:
-Cannot be used when celloidin embedding have to be done
-Highly inflammable
Isopropyl Alcohol (Isopropanol)
Dehydrating agents:
Advantages:
-Rapid dehydration within 1 hour.
-Miscible with water and organic solvents
-For clearing; can act as clearing agent
Disadvantages:
-Extreme volatile
-Highly inflammable
Acetone
Dehydrating agents:
Advantages:
-It is both dehydrating and clearing agent.
-Miscible with water, alcohol, xylene and paraffin.
Disadvantages:
-Overhardens the tissue if left for longer period
-It is expensive, toxic, and odorous
Dioxane
Dehydrating agents:
Advantage/s:
-Dehydrates rapidly and is not harmful to the tissues. The tissue may be transferred from water to normal saline directly to cellosolve and stored in it for months without producing hardening or distortion.
-Fixes and dehydrates tissues
Cellosolve
Dehydrating agents:
Advantage/s:
-Minimal distortion and hardening of tissues. Soluble in various clearing agents.
Disadvantage/s:
-Corrosive. Combustible. Strong irritant
Triethyl Phosphate
General Schedule for Alcohol Dehydration:
70% alcohol
6 hours
General Schedule for Alcohol Dehydration:
90% alcohol
12 hours
General Schedule for Alcohol Dehydration:
100%
2 hours, 1 hour, 1 hour
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 (De-alcoholization)
Clearing agents: (30mins to 1hr)
Most common
Advantage/s:
-It’s action is very rapid
-Cheap and slightly inflammable
-Readily eliminate in the paraffin oven
Disadvantage/s:
-Prolonged treatment with this reagent makes the soft tissues like brain and spleen quite brittle.
Xylene
Clearing agents: (15mins-60mins)
Advantage/s:
-It penetrates the tissues rapidly
-It causes minimum shrinkage
-It is a cheap clearing agent
Disadvantage/s:
-It is a carcinogen and potentially causes cancer
-It is a flammable liquid
-Causes aplastic anemia (permanent)
Benzene
Clearing agents: (6-24hrs)
Advantage/s:
-It is widely used for its hardening effect
-Ideal for hard and delicate tissues like bone marrow and brain
Disadvantage/s:
-Its action is slower than other clearing agents
-It can cause faintness if inhaled
Chloroform
Clearing agents: (1-2hrs)
Advantage/s:
-Tissues can be kept in this for a longer period
-Its action is similar to benzene but it is less toxic
Disadvantage/s:
-It causes irritation if accidentally inhaled or come into contact with skin or eyes
Toluene
Clearing agents: (2-3days)
Waxy/oily in nature
Advantage/s:
-It had a gentle action on the tissues
-It is the excellent clearing agent for tissues
Disadvantage/s:
-It is very expensive
-It slowly penetrates the tissue surface
Cedar Wood Oil
Clearing agents:
This is not normally utilized as a routine clearing agent but is rather recommended for clearing embryos, insects and very delicate specimens, due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening.
Used for research purposes only
Aniline Oil
Clearing agents:
This reagent causes minimum shrinkage of tissues. However, its quality is not guaranteed due to its tendency to become adulterated. Wax impregnation after clearing with clove oil is slow and difficult. Tissues become brittle, aniline dyes are removed, celloidin is dissolved. All of these, plus the expensiveness of the solution, makes it unsuitable for routine clearing purposes
Oldest clearant
Clove Oil
Clearing agents:
May be used in clearing tissues for embedding. Its properties are very similar to that of chloroform although it is relatively cheaper. Its disadvantage is the same as that of chloroform. It produces considerable tissue hardening and is dangerous to inhale on prolonged exposure due to its highly toxic effects
Causes faintness
Carbon Tetrachloride
Clearing agents:
superior to ordinary dehydrating and clearing agent due to its ability to perform two processes at the same time, thereby shortening the total processing time and allowing more time for fixation. It is non toxic but has an offensive odor and should be used in a well–ventilated room
Tetrahydrofuran
Clearing agents:
Slow-acting clearing agents that can be used when double embedding techniques are required.
Methyl Benzoate and Methyl Salicylate
