Dehydration, Clearing, Infiltration, and Embedding PDF

MLSBIO107 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUE Dehydration  This process is commonly carried out by immersing specimens in a series of ethanol (alcohol) solutions of increasing concentration until pure, water-free alcohol is reached.  Alcohol progressively replaces water in all the cel...

MLSBIO107 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUE Dehydration  This process is commonly carried out by immersing specimens in a series of ethanol (alcohol) solutions of increasing concentration until pure, water-free alcohol is reached.  Alcohol progressively replaces water in all the cells of the specimen.  Ethanol is miscible with water in all proportions so that the water in the specimen is progressively replaced by the alcohol.  Recommended volume of dehydrating agent should not be less than 10x of the sample  A series of increasing concentrations is used to avoid excessive distortion of the tissue.  A typical dehydration sequence for specimens not more than 4mm thick would be: 1. 70% ethanol 15 min 2. 90% ethanol 15 min 3. 100% ethanol 15 min 4. 100% ethanol 15 min 5. 100% ethanol 30 min 6. 100% ethanol 45 min At this point, all but a tiny residue of tightly bound (molecular) water should have been removed from the specimen. Dehydrating agents  30% Ethanol – Recommended for delicate tissues, particularly embryonic tissues.  ETHYL ALCOHOL (ETHANOL) - Recommended for routine dehydration of tissues. It is considered to be the best dehydrating agent because it is fast- acting, it mixes with water and many organic solvents, and it penetrates tissues easily  METHANOL - Toxic dehydrating agent; primarily used for blood and tissue films and for smear preparation.  BUTYL ALCOHOL (BUTANOL) - Used in plant and animal micro-techniques, it is a slow dehydrating agent, it produces less shrinkage compared to ethanol and it is recommended for tissues that do not require rapid processing.  ACETONE - Cheap, rapid-acting dehydrating agent utilized for the most urgent biopsies which it dehydrates in ½ to 2 hours.  4% PHENOL - May be used as an additive to 95% ethanol baths as part of the dehydration process, it acts as a softener for hard tissues such as tendons, nail, or dense fibrous tissue.  COMMONLY USED CLEARING AGENTS: ❑ A.K.A. DEALCOHOLIZATION (15-30MINS TO 1HR) 1. XYLENE (MOST COMMON) ❑ Removal of the dehydrating agent and replacement of 2. TOLUENE 3. BENZENE a fluid (clearing agent that is miscible with both the 4. CHLOROFORM dehydrating agent and embedding medium) 5. CEDARWOOD OIL 6. ANILINE OIL ❑ The use of clearing agents makes the tissues 7. CLOVE OIL “TRANSLUSCENT” 8. CARBON TETRACHLORIDE ❑ Most are flammable liquids ❑ Clearing agents have low boiling points ❑ Duration of clearing: over clearing may cause tissue brittleness ❑ For frozen sections: glycerine and gum syrup are used. No need for dealcoholisation (it will just make tissues transluscent)  5.1 XYLENE 5.3 BENZENE ❑ AKA Xylol 5.2 TOLUENE ✓Recommended for ❑ Colorless clearing agent; most commonly used in routine procedures urgent biopsies and for ❑ Rapid clearing time (15-30mins to 1hr) ✓ Substitute for routine purposes xylene or benzene ❑ Can be used for celloidin sections ✓Doesn’t make tissues ❑ For tissue sections with a thickness