Fixative Table

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Chapter 3: FIXATION AND FIXATIVES | 1 Chapter 3: FIXATION AND FIXATIVES | 1

I. ALDEHYDE FIXATIVES

FIXATIVE

FIXATIVE DESCRIPTION DESCRIPTION PURPOSE PURPOSE ADVANTAGES ADVANTAGES DISADVANTAGESDISADVANTAGES

Formaldehyde Formaldehyde (Formalin) (Formalin)



 10% conc. is widely used10% conc. is widely used 

 made from the oxidation ofmade from the oxidation of

methyl alcohol methyl alcohol   3737 – – 40% w/v 40% w/v soluble in soluble in water water 

 35%-40%35%-40% gas by weight gas by weight 

 buffered tobuffered topH 7pH 7

(phosphate buffer) (phosphate buffer) Fixation time:

Fixation time:24 HOURS24 HOURS



 Precipitates may bePrecipitates may be

removed by filtration or by removed by filtration or by addition of 10% methanol addition of 10% methanol



 Bleaching of tissues may beBleaching of tissues may be

prevented by changing the prevented by changing the fluid fixative every three fluid fixative every three months

months



 preservespreserves fatfat and and mucinmucin 

 preservespreserves glycogenglycogen 

 preservespreserves proteinsproteins 

 recommenderecommended d forfor nervous tissuenervous tissue

preparation preparation



 cheap, readily available, easy to cheap, readily available, easy to prepare,prepare,

relatively stable relatively stable



 compatible with many stainscompatible with many stains 

 does not overharden tissuesdoes not overharden tissues 

 penetrates tissues wellpenetrates tissues well 

 does not does not precipitate proteinsprecipitate proteins 

 does not make tissues brittledoes not make tissues brittle 

 colored tissue photographycolored tissue photography 

 allows frozen tissue sections to allows frozen tissue sections to be preparedbe prepared

easily easily



 DOESN’T REQUIRE WASHING OUTDOESN’T REQUIRE WASHING OUT 

 Used for mailing specimensUsed for mailing specimens



 Fumes are irritating to nose and eyesFumes are irritating to nose and eyes 

 Solution is irritating to Solution is irritating to skin (causes allergicskin (causes allergic

dermatitis on prolonged contact) dermatitis on prolonged contact)



 May produce considerable shrinking ofMay produce considerable shrinking of

tissues tissues



 Does not harden some Does not harden some cytoplasmiccytoplasmic

structures adequately structures adequately



 If unbuffered:If unbuffered: 

formalin reduces both basophilic andformalin reduces both basophilic and

eosinophilic staining of cells

eosinophilic staining of cells (acidity of(acidity of formic acid may be used when applying formic acid may be used when applying the silver impregnation technique of the silver impregnation technique of staining)

staining)



forms abundant brown pigment granulesforms abundant brown pigment granules

on blood containing tissues due to on blood containing tissues due to blackening of hemoglobin blackening of hemoglobin



 Prolonged fixation:Prolonged fixation: 

bleaching of specimen/loss of bleaching of specimen/loss of naturalnatural

colors colors



dispersing of fat from the tissues into thedispersing of fat from the tissues into the

fluid fluid



dissolution or loss ofdissolution or loss ofglycogenglycogen,, biurate ofbiurate of

sodium crystals

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Chapter 3: FIXATION AND FIXATIVES | 2 10% Formol-Saline  Simple microanatomical fixative  Made up of saturated formaldehyde (40% w/v) Fixation time: 24 HOURS at 35˚C (95˚F) 48 HOURS at 20-25˚C (65-77˚F)

 Recommended for fixation of central

nervous tissues & general post-mortem tissues for histochemical examinations

 penetrates and fixes tissues evenly

 preserves microanatomic and cytologic details

with minimum shrinkage and distortion

 large specimens may be fixed for a long time

provided that the solution is changed every three months

 preserves enzymes and nucleoproteins  demonstrates fats and mucin

 does not overharden tissues; facilitates

dissection of specimen

 ideal for most staining techniques (including

SILVER IMPREGNATION)

 allows natural color of tissue to be restored

upon immersion in 70% alcohol

Similar to formalin with the following additions:

 slow fixative (≥ 24 hours)

 tissues tend to shrink during alcohol

dehydration (this may be reduced by secondary fixation)

 Metachromatic reaction of amyloid is

reduced

 Acid dye stains less brightly than when fixed

with mercuric chloride

10% Neutral Buffered Formalin/

Phosphate-Buffered Formalin

 most common fixative

Formula: 3.5mg Sodium dihydrogen phosphate (anhydrous) 6.5mg Disodium hydrogen phosphate (anhydrous) 100ml 40% Formaldehyde 900ml Distilled Water

Fixation Time: 4 – 24 HOURS

 recommended for preservation and

storage of surgical, post-mortem and research specimen

 prevents precipitation of acid formalin

pigments on post-mortem tissue

 best fixative for tissues containing iron

pigments and for elastic fibers which do not stain well after Susa, Zenker or Chromate fixation

 requires no post-treatment after fixation, so

the tissue goes directly to 80% alcohol for processing

 longer to prepare; time-consuming  positivity of mucin to Periodic acid-Schiff

(PAS) stain is reduced

 may produce gradual loss in basophilic

staining of cells

 reactivity of myelin to Weigert’s iron

hematoxylin stain is reduced

 INERT TOWARDS LIPIDS, especially neutral

fats and phospholipids

Formal-Corrosive (Formal Sublimate)

Fixation time:3 – 24 HOURS

 formol-mercuric chloride solution is

recommended for routine post-mortem tissues

 penetrates small pieces of tissue rapidly  produces minimum shrinkage and hardening  excellent for many staining procedures

including SILVER RETICULUM METHODS

 brighten cytoplasmic and metachromatic

stains better than with formalin alone

 cytologic structures and blood cells are well

preserved

 no need for “washing out”; tissues can be

transferred directly from fixative to alcohol

 penetration is slow (tissue sections should

not be more than 1 cm thick)

 forms mercuric chloride deposits  does not allow frozen tissue sections to be

made

 inhibits the determination of the extent of

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Chapter 3: FIXATION AND FIXATIVES | 3

 fixes lipids; especially neutral fats and

phospholipids

Alcoholic Formalin (Gendre’s) Fixative

 post-fixation with

phenol-formalin for 6 hours or more can enhance immunoperoxidase studies on the tissues and in some cases, electron microscopy, if it is necessary at a later time to establish a diagnosis

 used to fix sputum since it

coagulates mucus

 fixation is faster (reduced to one-half)  can be used for rapid diagnosis since it fixes

and dehydrates at the same time (i.e. frozen section)

 good from preserving glycogen and for

micro-incineration technique

 produces gross hardening of tissues  causes partial lysis of RBCs

 preservation of iron-containing pigments is

poor

 formaldehyde foes not give as good a

morphological picture as glutaraldehyde

 it causes little cross-linking under usual

fixation conditions where low concentration of proteins are used ( glutaraldehyde is more effective at cross-linking)

Glutaraldehyde

 made up of two

formaldehyde residues, linked by three carbon chains

 acts similarly to

formaldehyde

 sometimes used for routine

light microscopy

 buffered glutaraldehyde,

followed by secondary fixation in osmium tetroxide is satisfactory for electron microscopy Fixation time: varies from ½ hour (for small specimens) to 2 hours (maximum

contribution)

 2.5% solution – small tissue

fragments and needle biopsies Fixation time: 2 – 4 HOURS

 4% solution – large tissues less than

4mm thick

Fixation time: 6-8 HRS up to 24 HRS

 more stable effects on tissues, giving a firmer

texture with better tissue sections; especially of nervous tissues

 preserves plasma proteins better  produces less tissue shrinkage  preserves cellular structures

 recommended for enzyme histochemistry and

electron microscopy

 more pleasant and less irritating to the nose  does not cause dermatitis

 more expensive  less stable

 penetrates tissues more slowly

 tends to make tissue (i.e. renal biopsy) more

brittle

 reduces PAS positivity of reactive mucin

(may be prevented by immersing

glutaraldehyde-fixed tissues in a mixture of concentrated glacial acetic acid and aniline oil)

PRECAUTIONS:

 specimen vial must be kept refrigerated

during fixation process

 swirling the vials ensures that the

specimen is in contact with the fresh solution at all times as it might change

II. METALLIC fixatives

A. Mercuric chloride

FIXATIVE DESCRIPTION PURPOSE ADVANTAGES DISADVANTAGES

Mercuric Chloride

 most common metallic

fixative

 recommended for renal tissues,

fibrin, connective tissues & muscles

 penetrates & hardens tissues rapidly and well  nuclear components are shown in fine detail

 penetrates poorly & causes marked shrinkage

(4)

 frequently used in sat. aq.

solutions of 5-7%

 widely used as a secondary

fixative, reacting with a number of amino acid residues & accompanied by spectroscopic changes, probably due to reactions with histidine residues

 compound solutions must

always be freshly prepared

 use of metallic forceps and

metallic caps should be avoided

 contact of fixative with

personal jewelries should be avoided

 precipitates all proteins

 great affinity to acid dyes and is preferred in

lieu of formaldehyde for cytoplasmic staining

 Trichome staining is excellent

 routine fixative of choice for preservation of

cell detail in tissue photography

 permits brilliant metachromatic staining of

cells

fixative agents (this may be counteracted by addition of acid)

 rapidly hardens the outer layer of the tissue

with incomplete fixation of the center ( thin sections should be made)

 penetration beyond the first 2-3mm is slow

(not more than 5mm thickness of tissues should be used)

 if left in the fixative for more than 1-2 days,

the tissue becomes unduly hard and brittle

 prevents adequate freezing of fatty tissues

and makes cutting of frozen tissue difficult

 causes considerable lysis of RBCs and

removes iron from hemosiderin

 INERT TO FATS AND LIPIDS

 leads to the formation ofblack granular

deposits in the tissues (may be removed by adding sat. iodine solution in 90% alcohol; iodine will be decolorized with absolute alcohol in the subsequent stages of dehydration)

 reduces the amount of demonstrable

glycogen

 compound solutions deteriorate rapidly upon

addition of glacial acetic acid to formalin

 extremely corrosive to metals

Zenker’s Fluid

 made up of mercuric

chloride stock solution to which glacial acetic acid is added to it just before use to prevent turbidity & formation of dark precipitate

 solutions must always be

freshly prepared

 tissues should be cut thin

(2-3mm) & hollow organs should be opened to promote complete penetration & fixation

 good general preservative for all

kinds of tissues

 gives excellent staining results  recommended for fixing small pieces

ofliver, spleen, connective tissue fibers & nuclei

 produces fairly rapid and even fixation  stock solutions keep well without

disintegration

 recommended for Trichrome staining  permits brilliant staining for nuclear &

connective tissue fibers

 compatible with most stains

 may act as a MORDANT to make certain

special staining reactions possible

 penetration is poor

 not stable after addition of acetic acid  prolonged fixation (for more than 24 hours

(will make tissues brittle and hard

 causes lysis of RBCs & removes iron

hemosiderin

 does not permit cutting of frozen sections  tendency to form mercuric pigment deposits

or precipitates (may be removed by de-zenkerization)

 Bring slides to water

 Immerse in Lugol’s iodine (5 min.)  Wash in running water (5 min.)

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Fixation time: 12 – 24 HOURS  Wash in running water (5 min.)

 Proceed with required water-soluble stain  tissue must be washed in running water for

several hours (or overnight) before processing

 insufficient washing may inhibit or interfere

with good cellular staining

Zenker-formol (Helly’s solution)

Fixation time: 12 – 24 HOURS

 excellent microanatomic fixative for

PITUITARY GLAND, bone marrow, spleen & liver

 penetrates and fixes tissues well  nuclear fixation & staining is better than

Zenker’s

 preserve cytoplasmic granules well

 similar to Zenker’s

 brown pigments are produced if tissues

(especially blood containing organs) are allowed to stay in the fixative for more than 24 hours due to RBC lysis ( may be removed by immersing the tissue in sat. alcoholic picric acid or sodium hydroxide)

Heidenhain’s Susa Solution

 excellent cytologic fixative  after using this fixative, the

tissue should be transferred directly to a high-grade alcohol (i.e. 96% or absolute alcohol) to avoid undue swelling of tissues caused by treatment with low-grade alcohol Fixation time:3 – 12 HOURS

 recommended mainly for TUMOR

biopsies (especially of the skin)

 penetrates and fixes tissues rapidly & evenly  produces minimum shrinkage and hardening

of tissues due to the counter-balance of the swelling effects of acids and the shrinkage effect of mercury

 permits most staining procedures to be done,

including silver impregnation, producing brilliant results with sharp nuclear & cytoplasmic details

 permits easier sectioning of large blocks of

fibrous connective tissues

 Susa-fixed tissues may be transferred directly

to 95% alcohol or absolute alcohol, thereby reducing processing time

 prolonged fixation of thick materials may

produce considerable shrinkage, hardening and bleeding (tissues should not be >1mm)

 RBC preservation is poor

 some cytoplasmic granules are dissolved  mercuric chloride tend to form in tissues

(these may be removed by immersion of tissues in alcoholic iodine solution )

 Weigert’s method of staining elastic fibers is

not possible in Susa-fixed tissues

B-5 Fixative

 prior to use, add 1cc. of

formaldehyde (40%) for 10cc. of B-5

Fixation time:1 ½ HR – 2 HRS

 used forBONE MARROW BIOPSIES  good fixative for cytology of bone marrow

biopsies

 overfixation hardens the tissue and makes

cutting difficult

 some B-5 solutions will form precipitate on

standing (but this is of no consequence)

 may causing mercuric pigments to form on

tissues (de-zenkerization may be employed)

B. CHROMATE FIXATIVES

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Chromic Acid

 used in 1-2% aq. solution,

usually as a constituent of a compound fixative

 adequately preserve carbohydrates

 it’s a strong oxidizing agent (a strong

reducing agent, such as formaldehyde, must be added to chrome-staining fixatives before use in order to prevent counteracting effects & consequent decomposition of solution upon prolonged standing)

Potassium Dichromate

 used in a 3% aq. solution  fixes but does not precipitate cytoplasmic

structures

 preserves lipids

 preserves mitochondria (if used inpH 4.5 –

5.2)

 if solution becomes acidified, cytoplasm,

chromatin bodies & chromosomes are fixed but mitochondria is destroyed

Redard’s (Muller’s Fluid)

Fixation time: 12 – 48

 recommended for demonstration of

chromatin, mitochondria, mitotic figures, Golgi bodies, RBC & colloid-containing tissues

 penetrates tissues well

 hardens tissues better and more rapidly than

Orth’s fluid

 deteriorates & darkens on standing due to

acidity (solution must always be freshly prepared)

 penetration is slow (tissues should be no

thicker than 2 – 3 mm)

 chromate fixed tissues tend to produce

precipitates od sub-oxide ( tissues should be thoroughly washed in running water prior to dehydration)

 prolonged fixation blackens tissue pigments

like melanin (tissues should be washed in running water prior to dehydration)

 glycogen penetration is poor; generally

contraindicated for carbohydrates

 nuclear staining is poor  does not preserve fats

 preserved hemosiderin less than buffered

formalin

 intensity of PAS reaction is reduced

Orth’s Fluid

• strong formaldehyde (40%) is added just before use Fixation time:36 – 72 HOURS

 demonstratesRICKETTSIAE & other

bacteria

 recommended for the study of degenerative

processes & tissue necrosis

 preserves myelin better than buffered

formalin

 same as Regaud’s fluid

C. LEAD FIXATIVES

Lead Fixatives used in 4% aq. solution of basic lead acetate

recommended for acid mucopolysaccharides fixes connective tissue mucin

takes up CO2 to from insoluble lead carbonate especially upon prolonged standing ( removed

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Chapter 3: FIXATION AND FIXATIVES | 7 by filtration or adding acetic acid drop by drop to lower the pH and dissolve the residue)

III. PICRIC ACID fixatives

Picric Acid

 Normally used in sat. aq.

solution (approx.. 1%)

 yellow dye may be removed

by treatment with another acid dye or lithium carbonate

 excellent fixative for glycogen

demonstration

 stable, penetrates tissues well & fixes small

tissues rapidly

 yellow stain prevents small fragments from

being overlooked

 allows brilliant staining with the trichrome

method

 suitable for Aniline stains (Mallory’s,

Heidenhain’sor Masson’s methods)

 dyes tissues yellow (dye may be removed by

treatment with another acid dye or lithium carbonate)

 excessive staining may be removed by

placing the tissues in 70% ethanol followed by 5% sodium thiosulfate & washed with running water

 causes RBC hemolysis & reduces amount of

demonstrable ferric iron in tissues

 not suitable for frozen sections because they

will crumble when cut

 prolonged fixation makes tissues hard, brittle

& difficult to section ( not longer than 12 – 24 hours; depending on size)

 picrates are formed upon protein & are soluble

in water (tissues must be rendered insoluble by direct immersion in 70% alcohol)

 fixed tissues must never be washed in water

prior to dehydration

 picric acid is highly explosive when dry (keep

moist with distilled water or sat. alcohol at 0.5 to 1% conc. during storage)

 alters and dissolves lipids

 interferes with Azure eosin method of staining

(tissues should be thoroughly washed with alcohol)

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Bouin’s Solution

 fc

Fixation time: 6 – 24 HOURS

 recommended for fixation of

embryos & pituitary biopsies

 produces minimal distortion of

microanatomical structures

 can be used for general & specific stains

(shrinking effect of picric acid is balanced by the swelling effect of glacial acetic acid)

 excellent fixative for preserving soft &

delicate structures (i.e. endometrial curettings)

 penetrates rapidly & evenly, causing little

shrinkage

 yellow stain is useful when handling

fragmented biopsies

 permits brilliant staining of tissues

 preferred fixative for tissues to be stained by

Masson’s trichrome forcollagen, elastic or connective tissue (if tissue is fixed in formalin, a pre-treatment of Bouin, as a mordant prior to trichrome stain, is recommended)

 preserves glycogen

 does not need “ washing out”

 penetrates large tissues poorly

 picrates are soluble in water (tissues must be

transferred directly from fixative to 70% alcohol)

 not suitable for fixing kidney structures, lipids

& mucus

 destroys cytoplasmic structures (i.e.

mitochondria)

 produces RBC hemolysis & removes

demonstrable ferric iron from blood pigments

 reduces or abolishes Feulgen reaction due to

hydrolysis of nucleoproteins

Brasil’s Alcoholic Picroformol

Fixative

 better and less messy than Bouin’s solution  excellent fixative for glycogen

GLACIAL ACETIC ACID

Acetic Acid

colorless liquid

called “Glacial” Acetic Acid

when undiluted because it is a water-free (anhydrous) acetic acid that freezes and solidifies at about 17°C.

 Normally used in conjunction with

other fixatives to form a compound solution

 It precipitates DNA, which is split off

from nucleoprotein

 valuable for the preservation of

nuclei

It fixes and precipitates nucleoproteins. It precipitates chromosomes and chromatin

materials; hence, is very useful in the study of nuclear components of the cell. In fact, it is an essential constituent of most compound nuclear fixatives.

It causes tissues (especially those containing

collagen) to swell. This property is used in certain compound fixatives to counteract the shrinkage produced by other components (e.g. mercury).

When combined with Potassium Dichromate,

the lipid-fixing property of the latter is destroyed (e.g. Zenker's fluid).

It is contraindicated for cytoplasmic fixation

since it destroys mitochondria and Golgi elements of cells.

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Alcohol fixatives

Methyl Alcohol 100%

It is excellent for fixing dry and wet smears,

blood smears and bone marrow tissues.

It fixes and dehydrates at the same time.

Penetration is slow.

If left in fixative for more than 48 hours,

tissues may be over hardened and difficult to cut.

Isopropyl Alcohol 95%

 is used for fixing touch preparations

 some touch preparations are air

dried and not fixed, for certain special staining procedures such as Wright-Giemsa

Ethyl Alcohol

is used at concentrations of

70-100%

use of lower concentrations

cause hemolysis of RBC and inadequately preserved WBC Fixation Time: 18-24 HOURS

It may be used as a simple fixative. frequently incorporated into

compound fixatives for better results

Preserves but does not fix glycogen. Fixes blood, tissue films and smears. Preserves nucleoproteins and nucleic acids;

used for histo-chemistry, especially for enzyme studies.

Fixes tissue pigments fairly well.

Causes polarization of glycogen granules Produces considerable hardening and

shrinkage of tissues

Hemosiderin preservation is less than in

buffered formaldehyde

A strong reducing agent; should not be mixed

w/ strong oxidizing agents such as chromic acid, potassium dichromate& osmium tetroxide

Carnoy’s Fluid

Rapid in action

Tissues fized in Carnoy’s for 1

hr can be transferred directly to absolute alcohol, or an alcohol-chloroform mixture (1:1)

Fixation Time: 1-3 HOURS

Recommended for fixing

chromosomes, lymph glands & urgent biopsies

May be used for urgent biopsy spx for

paraffin processing w/in 5 hrs

Used to fix brain tissues for diagnosis

of rabies

Most rapid fixative; may be used for urgent

biopsy specimens for paraffin processing w/in 5 hrs

Fixes and dehydrates at the same time. Permits good nuclear staining and

differentiation.

Preserves Nissl granules and cytoplasmic

granules well.

Preserves nucleoproteins and nucleic acids. Excellent fixative for glycogen since aqueous

solutions are avoided.

Very suitable for small tissue fragments such

as curettings and biopsy materials.

Following fixation, tissues may be transferred

directly to absolute alcohol, thereby shortening processing time.

Produces RBC hemolysis

Causes considerable tissue shrinkage Suitable ONLY for small pieces of tissues due

to slow penetration

Harden tissues excessively & distorts tissue

morphology

Dissolves fat, lipids, myelin

Leads to Polarization (unless very cold temp

[-70C] are used)

Dissolves acid-soluble cell granules and

pigments

Newcomer’s Fluid

Fixation Time: 12-18 HOURS @ 3°C

Rec for fixing mucopolysaccharides and

nuclear proteins.

Produces better reaction in Feulgen stain than

(10)

Acts both as a nuclear and histochemical

fixative.

Osmium tetroxide (osmic acid)

Flemming’s Solution

Most common

chromium-osmium acetic acid fixative used

Fixation Time: 24-48 HOURS

Recommended for nuclear

preparation of such sections

It is an excellent fixative for nuclear

structures, e.g. chromosomes.

It permanently fixes fat.

Relatively less amount of solution is required

for fixation (less than 10 times the volume of the tissues to be fixed).

It is a poor penetrating agent; hence, is

applicable only to small pieces of tissues.

The solution deteriorates rapidly and must be

prepared immediately before use.

Chromic-osmic acid combinations depress the

staining power of hematoxylin (especially Ehrlich's hematoxylin).

It has a tendency to form artifact pigments;

these may be removed by washing the fixed tissue in running tap water for 24 hours before dehydration.

It is very expensive.

Flemming’s Soluton w/o

acetic acid

Made up of chromic and

osmic acid

Removal of acetic acid from

the formula serves to improve the cytoplasmic detail of the cell

Recommended for cytoplasmic

structures particularly the mitochondria

Same as Flemming’s Same as Flemming’s

Trichloroacetic acid

Tricholoracetic Acid

reagent that is used for the

precipitation of proteins and nucleic acids

Also used as a decalcifier and fixative

in microscopy.

Sometimes incorporated into

compound fixatives

Precipitates proteins.

 Its marked swelling effect on tissues serves to

counteract shrinkage produced by other fixatives.

May be used as a weak decalcifying agent Its softening effect on dense fibrous tissues

facilitates preparation of such sections.

Poor penetrating agent; suitable only for

small pieces of tissues or bones

Acetone

(11)

Chapter 3: FIXATION AND FIXATIVES | 11 Acetone Used at ice cold temperature

ranging from -5°C to 4°C

Fixation time may vary from

several minutes (for cell smears, cryostat sections) to several hours (1-24 hours for small tissue blocks).

Its use is reserved for the fixation of

cryostat sections or for tissues in which enzymes have to be preserved.

It is recommended for the study of water

diffusible enzymes especially phosphatases and lipases.

It is used in fixing brain tissues for diagnosis of

rabies.

It is used as a solvent for certain metallic salts

to be used in freeze substitution techniques for tissue blocks.

Produces inevitable shrinkage and distortion Dissolves fat

Preserves glycogen poorly Evaporates rapidly

Heat fixation

Involves thermal coagulation

of tissue proteins for rapid diagnosis

Usually employed for frozen tissue

sections and preparation of bacteriologic smears

Fixation is better

Preserves nuclear and cytoplasmic detail Suitable for frozen tissues preparation

Produces considerable tissue shrinkage and

distortion

Destroys RBC