3.dehydration ht

dehydration

Dehydration first step in processing Water is present in tissues in free and bound

(molecular) forms removing some or all of the free water cellular components are dissolved by

dehydrating fluids certain lipids are extracted by anhydrous

alcohols water soluble proteins are dissolved in the

lower aqueous alcohols

Dehydration necessary in all infiltration methods, except where

tissues are simply externally supported by an aqueous embedding medium

Choice of a dehydrant is determined by the nature of the taskthe embedding medium processing methodeconomic factors.

Dehydrants differ in their capacity to cause tissue shrinkage

Principles of Dehydration Dilution dehydration

the most commonly used method increasing concentrations of hydrophilic or water

miscible fluidseventually replace free water in the tissues

Chemical dehydrationthe dehydrant is hydrolysed by free water present in

tissues to form acetone and methanol in an endothermic reaction

acidified dimethoxypropane or diethoxypropane

Procedures paraffin wax method

initiated in 60%-70% ethanolprogressing through 90%-95% ethanol2 or 3 changes of absolute ethanol before

proceeding to the clearing stage

NB: well fixed tissues can be transferred directly to

95% ethanol incompletely fixed tissues may exhibit artifacts

if placed directly in higher alcohols

Procedures

NB:The dehydrant concentration at which

processing is initiated depends largely upon the fixative employed

To minimize tissue distortion from diffusion currents, delicate specimens are dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100% ethanol

Procedures

NB:Duration of dehydration should be kept to

the minimum consistent with the tissues being processed

Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol

blocks 5 mm thick require up to 90 minutes or longer in each change

Tissues may be held and stored indefinitely in 70% ethanol without harm.

Characteristics of an ideal dehydrating solution:

It must dehydrate rapidly without producing considerable shrinkage or distortion of tissues

It should not evaporate fast It should be able to dehydrate even fatty

tissues It should not harden tissues excessively

Characteristics of an ideal dehydrating solution:

It should not remove stains It should not be toxic to the body It should not be a fire hazard

TYPES OF DEHYDRANTS

ALCOHOLSalso act as secondary coagulant

fixativesEthanol (+Anhydrous cupric sulfate) MethanolIsopropanol Normal and tertiary butanols

TYPES OF DEHYDRANTS

GLYCOL-ETHERSdo not act as 2º fixatives only as solventsdo not appear to alter tissue reactivity.

2-Ethoxyethanol, ethylene glycol monoethyl ether, cellosolve or oxitol

Ethoxyethanol Dioxane, 1,4 diethylene dioxide Polyethylene glycols (PEG)

TYPES OF DEHYDRANTS

OTHER DEHYDRANTSAcetone (best dehydrant for fatty spx) Tissues

are dehydrated through four changes of acetone, the last of which should always be fresh.

Tetrahydrofuran 2,2 dimethoxypropane (DMP) and 2,2

diethoxypropane (DEP) Phenol, beechwood creosote and aniline

Advantages & Disadvantages

See table

Dehydrating Agents

A. ALCOHOL clear, colourless, flammable, hydrophilic

liquids miscible with water and with most organic

solvents (anhydrous) also act as secondary coagulant fixatives

during tissue processing

Dehydrating agentsA. ALCOHOLA.1. Ethanol

probably the most commonly used dehydrant in histology

supplied as 99.85% ethanol (absolute ethanol, 100 High Grade or Standard Grade)

as special Methylated Spirits (99.85% ethanol denatured with 2% methanol)

Both are satisfactory for histological purposes

Dehydrating agentsA.1.Ethanol

rapid, efficient and widely applicable dehydrant

normally a poor lipid solvent except under microwave processing conditions

dissolves nitrocellulose slowly unless combined in equal proportions (or better, 1:2)with diethyl ether

Processing times in absolute ethanol should be minimal

Dehydrating agentsA.1.Ethanol

Progressive removal of bound water from carbohydrates and proteins during prolonged immersion in absolute ethanol causes tissues to harden excessively and become brittle

Colloid, blood, collagen and yolky tissues are particularly affected

The problem is exacerbated by heat during wax infiltration.

Dehydrating agentsA.1. Ethanol

Anhydrous cupric sulphate added to the final absolute ethanol on a tissue processor scavenges any water present

The salt is self-indicating: white when anhydrous, blue when hydrated, and is only slightly soluble in ethanol

Anhydrous calcium sulphate (Drierite) or molecular sieves act in a similar manner but are non-indicating

Dehydrating agentsA.2. Methanol

is a good ethanol substitute but rarely used for routine processing because of its volatility, flammability and cost

It is a poor lipid solvent, and will not dissolve nitrocellulose unless mixed with acetone

In microwave processing it tends to harden tissues more than ethanol

Dehydrating agentsA.3. Isopropanol

It is a universal solvent available as 99.8% (absolute) isopropanol

slightly slower in action and not as hygroscopic as ethanol, but a far superior lipid solvent

completely miscible with water and most organic solvents

is fully miscible with melted paraffin wax and is readily expelled from tissues and wax baths


shrinks and hardens tissues less than ethanol

used to dehydrate hard, dense tissues, which can remain in the solvent for extended periods without harm

To minimize shrinkage, fixed tissues are transferred via 60%-70% isopropanol or ethanol to absolute isopropanol.


Isopropyl alcohol has also been recommended as a xylene substitute

unsatisfactory as a dehydrant in microwave stimulated processing

used as a transition solvent following ethanol dehydration

only dissolves nitrocellulose in the presence of esters such as methyl benzoate or methyl salicylate


cannot be used as a dehydrant in alcohol-ether-celloidin techniques

solvent for some lipid-soluble dyes, but is not used in staining work stations as many other dyes are insoluble in this solvent

Dehydrating agentsA.4. Normal and tertiary butanols

are universal solvents mainly used for small-scale manual processing of plant and animal tissues in teaching and research

Normal butanol is recommended for processing lightly chitinised arthropods and rodent tissues It causes less hardening and shrinkage than

ethanol though this is offset by the prolonged processing schedules which may result in tissue shrinkage


N-butanol is poorly miscible with water and only

slowly miscible with paraffin wax It is flammable, with a penetrating

camphor-like odor, and the vapors are eye irritants

Iso-butanol, with similar properties and processing characteristics is a less costly substitute for n-butanol


Tertiary-butanol is widely used in plant histology but rarely for animal tissues a major disadvantage when below 26°C

it is hygroscopic crystalline solid In processing it is used in a similar

manner to n-butanol

dehydrating agentsB. GLYCOL-ETHERS do not act as secondary fixatives do not appear to alter tissue reactivity.

B.1. 2-Ethoxyethanolethylene glycol monoethyl ether,

cellosolve or oxitolused as a dehydrant preceding polyester

wax embeddingfor dehydration following dioxane-based

fixation of hard animal tissues in the agar-ester wax double embedding

technique

dehydrating agentsB.1. 2-Ethoxyethanol

colorless, nearly odorless flammable liquidstrongly hygroscopic, miscible with water

and most organic solventsrapid but non-hardening in action, and

tissues can remain in it for yearsTo avoid severe shrinkage, tissues are

transferred from aqueous fixative or washing via 60%-70% ethanol into full strength cellosolve.

dehydrating agentsB.2. Dioxane

1,4 diethylene dioxide causes less tissue shrinkage and hardening than ethanol

excellent for tissues excessively hardened by ethanol-xylene processing

has a rapid but gentle action, and is best used in a graded series

Tissues may remain in it for long periods without harm


colorless, flammable universal solvent with an odor similar to butanol

freezes at 12°C, and is miscible with water, most organic solvents and paraffin wax

dissolves mercuric chloride, but precipitates potassium dichromate and other salts

It is cumulatively toxic and a suspected carcinogen


expensive and is normally reclaimed by drying over a 10-20 mm layer of calcium oxide or anhydrous cupric sulphate

also recovered by freezing hydrated solvent in a spark-proofed refrigerator at 2-5°C

Water, which separates out, is decanted from the crystalline dioxane which is then thawed, finally dried over a solid dehydrant and reused

Explosive peroxides form in dioxane exposed to air

dehydrating agents

B.3. Polyethylene glycols (PEG) water miscible polymers used to dehydrate

and embed substances labile to the solvents and heat of the paraffin wax method

clear, viscous, slightly hydroscopic liquids or solids of low toxicity

miscible with most organic solvents and dissolve nitrocellulose

Dehydration is initiated in the low molecular weight liquid glycols

dehydrating agents

B.3. Polyethylene glycols (PEG) low Tissues pass through glycols of

increasing molecular weight and viscosity, and are finally embedded in a high molecular weight PEG which is solid at room temperature

Polyethylene glycol used for dehydration can be regenerated by heating at 104°C for 24 hours

dehydrating agents

C. ACETONE colorless flammable liquid with sharp

characteristic ketonic odor low toxicity and is freely miscible with water

and organic solvents It is a fast, effective dehydrant though it may

cause tissue shrinkage it may also act as a coagulant secondary

fixative

dehydrating agents

C. ACETONE the best dehydrant for processing fatty

specimensTissues are dehydrated through four

changes of acetone, the last of which should always be fresh

Acetone is not recommended for microwave processing as it causes excessive nuclear shrinkage

dehydrating agents

D. TETRAHYDROFURAN colorless, highly volatile and flammable

universal solvent with an offensive ethereal odour

completely miscible with water, most organic solvents, paraffin wax and mounting media

dissolves mountants, but not most dyes dehydrates rapidly causing little shrinkage or

hardening, and is possibly the best of the universal solvents

dehydrating agents

D. TETRAHYDROFURAN less toxic than dioxane for which it can be

substituted Tissues are processed as in dioxane method form explosive peroxides which renders

solvent recovery distillation dangerous66.

dehydrating agents

E. 2,2 dimethoxypropane (DMP) and 2,2 diethoxypropane (DEP)

used for chemical dehydration of tissues flammable and form peroxides miscible with paraffin wax however methanol,

one of the hydrolysis products, is not wax miscible and a post dehydration rinse in acetone

a transition solvent such as methyl salicylate or toluene should precede infiltration with wax

dehydrating agents

E. 2,2 dimethoxypropane (DMP) and 2,2 diethoxypropane (DEP)

shrinks tissues slightly less than DEP Chemical dehydration is suitable for rapid

manual processing or machine processing, and is comparable to conventional dehydration for tissue morphology and staining reactions

Acidified DMP/DEP can be reused several times, though dehydration times need to be extended. The reagent is stored at 4°C in a spark-proofed refrigerator

dehydrating agents

F. PHENOL beechwood creosote and aniline facilitate

dehydration when mixed with transition solvents

The coupling action permits tissues and celloidin sections to be cleared from lower strength alcohols

Creosote and aniline are used less commonly though in a similar manner to phenol

dehydrating agentsF. PHENOL Phenol consists of clear hygroscopic acicular

crystals and is also available as 80% w/w liquefied phenol

it is soluble in water, alcohol and most organic solvents except petroleum ethers

Concentrated solutions coagulate nitrocellulose On exposure to air and light, phenol and its solutions

develop a pink to reddish discolouration. Containers must be protected from light and tightly sealed

Phenol crystals and 80% concentrate react violently with formaldehyde.

finish

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3.dehydration ht

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