This document discusses tissue dehydration techniques used in histology sample preparation. It describes the ideal characteristics of dehydrating solutions and lists commonly used agents such as alcohols, acetone, dioxane, cellosolve, triethylphosphate, and tetrahydrofuran. Details are provided on the properties and procedures for each agent, including advantages like rapid dehydration and disadvantages like toxicity. The goal of dehydration is to remove water from tissues prior to wax impregnation while minimizing shrinkage, distortion, and other tissue damage.
This document discusses tissue dehydration techniques used in histology sample preparation. It describes the ideal characteristics of dehydrating solutions and lists commonly used agents such as alcohols, acetone, dioxane, cellosolve, triethylphosphate, and tetrahydrofuran. Details are provided on the properties and procedures for each agent, including advantages like rapid dehydration and disadvantages like toxicity. The goal of dehydration is to remove water from tissues prior to wax impregnation while minimizing shrinkage, distortion, and other tissue damage.
This document discusses tissue dehydration techniques used in histology sample preparation. It describes the ideal characteristics of dehydrating solutions and lists commonly used agents such as alcohols, acetone, dioxane, cellosolve, triethylphosphate, and tetrahydrofuran. Details are provided on the properties and procedures for each agent, including advantages like rapid dehydration and disadvantages like toxicity. The goal of dehydration is to remove water from tissues prior to wax impregnation while minimizing shrinkage, distortion, and other tissue damage.
1. Should dehydrate rapidly without producing considerable shrinkage or distortion of tissues 2. Should not evaporate very fast 3. Should be able to dehydrate even fatty tissues 4. Should not harden tissues excessively 5. Should not remove stains 6. Should not be toxic to the body 7. Should not be a fire hazard Commonly used Dehydrating Agents: 1. Alcohol 2. Acetone 3. Dioxane 4. Cellosolve 5. Triethylphosphate 6. Tetrahydrofuran I. ALCOHOL Ethyl alcohol (ethanol) – recommended for routine dehydration - clear, colorless, flammable fluid - Best dehydrating agent (fast acting, mixes with water and many organic solvents, penetrates tissues easily) - Not poisonous - Not very expensive Methyl alcohol – for blood and tissue films; smear preparations - Toxic dehydrating agent Butyl alcohol – utilized in plant and animal micro-techniques - Slow dehydrating agent – produces less shrinkage and hardening than ethanol - recommended for tissues which do not require rapid processing Tissues is passed through a series of progressively increasing concentrations of alcohol Temperature of 37°C – hasten dehydration time For complete dehydration: ¼ in deep of anhydrous copper sulfate is placed at the bottom of the container and covered with filter paper (accelerate dehydration by removing water from dehydrating fluid) - Blue discoloration of copper sulfate crystals will indicate full saturation of dehydrating fluids with water - Alcohol is discarded and changed with a fresh solution ACETONE Cheap Rapid acting dehydrating agent (dehydrates in ½ to 2 hours) - Penetrates tissues poorly Clear, colorless fluid – mixes with water, ethanol and most organic solvents More miscible with epoxy resins than alcohol Highly flammable – requires considerable care in handling Causes brittleness in tissues if placed for prolonged period of time Most lipids are removed from tissues Use is limited to small pieces of tissues due to extreme volatility and inflammability Not recommended for routine dehydration purposes – due to tissue shrinkage Excellent dehydrating and clearing agent Readily miscible in water, melted paraffin, alcohol and xylol Produces less tissue shrinkage compared to alcohol dehydration Tissues can be left for long period of time without affecting the consistency or staining properties Tend to ribbon poorly Expensive Extremely dangerous (main disadvantage) - Vapor produces a cumulative and highly toxic action in man - Not recycled as risk of creating explosive peroxides increases Two methods of dehydration : - Graupner’s Method: 1st pure dioxane soln --------------------- 1 hour 2nd pure dioxane soln -------------------- 1 hour 3rd pure dioxane soln -------------------- 2 hours 1st paraffin wax ---------------------------- 15 minutes 2nd paraffin wax --------------------------- 45 minutes 3rd paraffin wax --------------------------- 2 hours Embed in mold and cool in water - Weiseberger’s Method – tissue is wrapped in a gauze and suspended in a bottle containing dioxane and a little anhydrous calcium oxide - water is displaced from the tissue and absorbed by calcium oxide or quick lime - dehydration period : 3 – 24 hours (ETHYLENE GLYCOL MONOETHYL ETHER) Dehydrates rapidly Tissue may be transferred from water or normal saline to cellosolve and store it for months without hardening or distortion Ethylene glycol ether – combustible at 110 – 120°F - Toxic by inhalation, skin contact and ingestion - Following exposure, the reproductive ,fetal, urinary and blood systems are particularly vulnerable to toxic side effects Removes water very readily and produces very little distortion and hardening of tissue Soluble in water, alcohol, ether, benzene, chloroform, acetone and xylene Dehydrate sections and smears following certain stains and produces minimum shrinkage Both dehydrates and clears tissues Miscible in both water and paraffin Dissolve may substances including fats Miscible in lower alcohols, ether, chloroform, acetone, benzene, and xylene Used for demixing, clearing and dehydrating paraffin sections before and after staining Causes less shrinkage and easier cutting of sections with fewer artifacts Does not dissolve aniline dyes Toxic if ingested or inhaled - Vapors cause nausea, dizziness, headache - Eye and skin irritant – prolonged exposure (6 mons) cause conjuctival irritation - no practical way of absolutely protecting the skin against contact – use of THF should be avoided Additives to Dehydrating Agents: - 4% phenol added to 95% ethanol baths – acts as softener for hard tissues - Immersed in glycerol/alcohol mixture - Molliflex