process where the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all cavities.

aka impregnation

aka Casting or Blocking

impregnated tissue is placed into precisely arranged position in a mold containing the medium, which then allowed to solidify.

also termed as De-alcoholization; Alcohol or a dehydrating agent is removed from tissues. Replaced with a substance that will dissolve the wax with which the tissue is to be impregnated (Paraffin) or the medium on which the tissue is to be mounted (Canada Balsam

t issue is placed in the mold containing the medium. This is allowed to solidify.

- Tissues are immersed in the melted paraffin at a temperature of 5-10 Celsius above the melting point.

- Cooled rapidly in a refrigerator at – 5 Celsius or immersed in cold water to solidify.

- Orientation is the accurate placing of a tissue in the mold during this process or on a microtome before cutting.

- The surface of the tissue that will be examined is the part that must be placed parallel to the bottom of the mold.

Used for routine dehydration of tissues

For routine dehydration of tissues; the best dehydrating agent; Non-toxic and not very expensive

Which of the following dehydrating agents is toxic and used for blood and tissue films?

Which of the following dehydrating agents is used in plant and animal micro-techniques; reccomended for tissues that do not require rapid processing?

Diethylene Dioxide

Cheap, rapid dehydrating agent used for most urgent biopsies; Most lipids are removed from the tissues. However, this is extremely volatile and flammable. Not recommended as a routine dehydrating agent

- Excellent dehydrating and clearing agent readily miscible in water, melted paraffin, alcohol and xylol.

- Tissues may be placed directly into this agent right after washing out, but may tend to ribbon poorly.

- Extremely dangerous (Vapors produce a cumulative and highly toxic action in man)

Ethylene Glycol Monoethyl Ether

- Reproductive, Fetal, Urinary and Blood systems are particularly vulnerable to the toxic side effects

- Reproductive, Fetal, Urinary and Blood systems are particularly vulnerable to the toxic side effects

Ethylene Glycol Monoethyl Ether

- Propylene – based glycol ethers should be used instead of ethylene based glycols.

- Soluble in water, alcohol, benzene, ether, chloroform, acetone and xylene

- a reagent that both dehydrates and clears tissues; miscible in both water and paraffin

- Dissolves fat, also miscible in lower alcohols, ether, chloroform, acetone, benzene and xylene

- Used for demixing, clearing and dehydrating paraffin sections before and after staining.

- Doesn’t dissolve aniline dyes

- Toxic when inhaled or ingested. Vapors cause dizziness, headache and anesthesia.

- It is an eye irritant, prolonged exposure like in 6 months may cause conjunctival irritation.

What is the concentration of Phenol added to 95% Ethanol baths that gives softening effect to hard tissues like tendons, nails, or dense fibrous tissues?

Hard tissues can be placed in a Glycerol/Alcohol mixture or in Molliflex

Xylene

Dioxane

Chloroform and Cedarwood

Glycerin and Gum Syrup

Colorless, commonly used in the histopathology lab. Usually has ½ to 1 hour clearing time

- Used also for embedding and mounting procedures

Suitable for processing schedules of less than 24 hours and when tissue block is less than 5mm.

- Miscible with Absolute Alcohol and Paraffin

- Doesn’t dissolve Celloidin and doesn’t extract Aniline Dyes

- Not for nervous tissues and lymph nodes

- Clearing agent preferred in embedding process of tissue.

- For urgent biopsies (15-60 minutes)

- Volatilizes rapidly in paraffin oven

- Miscible with absolute alcohol only

- Carcinogenic, May damage Bone Marrow (Aplastic Anemia)

Substitute for Xylene and Benzene for clearing both during embedding and mounting process. Time recommended is 1- 2 hours.

- Not carcinogenic.

- High concentrations emit fumes that are toxic upon prolonged exposure.

- Slower in action than Xylene

- Thicker tissue blocks can be cleared. (up to 1 cm)

- Tissue placed in this clearing agent doesn’t make it translucent.

- for tough tissues like the skin, fibroid and decalcified tissues

- for nervous tissue, lymph nodes and embryos

- toxic to the liver after prolonged inhalation

- Wax impregnation is slow.

- May attack the rubber seal used in vacuum impregnating bath

- Clears for both paraffin and celloidin sections during embedding process.

Recommended for Central Nervous system and cytological studies. (Smooth muscles and the skin)

- Requires two changes of clearing solutions.

- Miscible with 96% Alcohol which it removes readily.

- Clears Celloidin in 5-6 Days

- Improves cutting of the sections.

- Doesn’t dissolve aniline Dyes

- Becomes milky upon prolonged storage and should be filtered before use

Wax impregnation after clearing with this clearing agent is slow and difficult

- Aniline Dyes are removed.

- Tissue becomes brittle. Celloidin is dissolved.

- Not normally utilized as a routine clearing agent.

- Clearing tissues for embedding

- Similar to chloroform

- Recommended for clearing embryos, insects and very delicate specimens

for skin and smooth muscles

for skin only

Artificial oil, for delicate tissues

for smooth muscles; has foul odor

for friable tissues

for the eyes

– for smooth muscles

Excellent Clearing agent

- Paraffin Wax with a melting point of 54-58 Celsius

- Paraffin Wax with a melting point of 50-54 Celsius

True about manual processing for paraffin impregnation

Four changes of wax are required at 15 minutes intervals in order to insure complete removal of the clearing agent

- Use of tissue processing machine (Autotechnicon, Elliot Bench Type Processor)

- 12 individual processing steps.

- Machine is mounted on rollers

- 10 One-liter capacity glass beakers and 2 thermostatically controlled wax baths.

- A transfer arm is electronically controlled from one processing reagent to another (By clock schedules)

- Constant agitation of fluid is accompanied by a continuous vertical movement

Precautions - Presence of Odor in the clearing agent – Paraffin wax must be changed. - Dehydrating agents must be changed frequently - The First 100% Ethanol must be discarded. Others moved down, so the final batch has the fresh 100% Ethanol after two complete processing runs of loads of at least three-quarters capacity. - Clearing agent and the dilute ethanol should be changed at least once a week - Wax bath thermostat should be set at least 3 degrees above the melting point of the wax

Involves wax impregnation under negative atmospheric pressure inside an embedding oven over to hasten the removal of air bubbles and the clearing agent.

- Removal of air bubbles particularly in the lungs

- Recommended for urgent biopsies

For delicate tissues like the brain, lungs, connective tissues decalcified bones, eyes, spleen and the Central Nervous system

- Vacuum chamber is maintained at 2-4 Celsius above the melting point of wax.

- Degree of vacuum should not exceed 500mm Hg.

- This technique provides the fastest impregnation.