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Types of Fixative

I. According to COMPOSITION

A.SIMPLE FIXATIVES are made up of only one

component substance

1.Aldehydesa.Formalin

b.Glutaraldehyde

2.Metallic Fixatives

a.Mercuric chloride

b.Chromate fixatives

i.Potassium dichromate

ii.Chromic acid

c.Lead fixatives

d.Picric acid

e.Acetic acid

f. Alcohol

B.COMPOUND FIXATIVES made up of two or

more fixatives which have been added together

to obtain the optimal combined effect of their

individual actions upon the cells or tissue

constituents.

II. According to ACTION

A.MICROANATOMICAL FIXATIVES those that

permit the general microscopic study of tissue

structures without altering the structuralpattern of the tissue in question.

1. 10% Formol saline

2. 10% Neutral buffered formalin

3. Heidenhains Susa

4. Formol sublimate

5. Brasils solution

6. Zenkers solution

7. Bouins solution

8. Zenker-formol

B.CYTOLOGICAL FIXATIVES those that

preserve specific parts and particular

microscopic elements of the cell itself3 Types of Cytological Fixatives

o Nuclear fixative

o Cytoplasmic fixative

o Histochemical fixative

1.NUCLEAR FIXATIVES those that preserve the

nuclear structure

Usually contain GLACIAL ACETIC ACID as a

primary component due to its affinity for

nuclear chromatin

Have pH of 4.6 or less

Examples:

o Flemmings fluid

o Bouins fluid

o Heidenhains Susa

o Carnoys fluid

o Newcomers fluid

2.CYTOPLASMIC FIXATIVES those that preserve

cytoplasmic structures.

Must never contain glacial acetic acid which

destroys mitochondria and Golgi bodies

Have a pH of more than 4.6

o Flemmings fluid w/o acetic acid

o Kellys fluid

o Formalin with post-chroming

o Regauds fluid (Mullers fluid)

o Orths fluid

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3.HISTOCHEMICAL FIXATIVES those that preserve the

CHEMICAL constituents of the cells and tissues.

Formol Saline 10%

Absolute Ethyl Alcohol

Newcomers fluid

Fixation of tissue can be accomplished by:

a.PHYSICAL METHOD

Heating

Freeze-drying

Microwaving

b.CHEMICAL METHOD

Coagulant fixatives

Compound fixatives

Cross-linking fixativesPHYSICAL METHODS

A.HEAT FIXATION

Simples form of fixation

Each component s less soluble in water after

heat fixation

Primarily used to accelerate other forms of

fixation as well as the steps of tissue

processing

B.MICROWAVE HEATING

Speeds up the fixation and can reduce times

for fixation of some gross specimens and

histological sections from more than 12 hours

to less than 20 mins.

Commercial glyoxal-based fixatives which do

not form vapours when heated at 55C

introduced as an efficient method of microwave

fixation.

C.FREEZE-DRYING and FREEZE SUBSTITUTION

Useful technique in studying SOLUBLE

MATERIALS and SMALL MOLECULES

Tissues are cut in small sections, immersed in

liquid nitrogen and the water is removed in avacuum chamber at -40C.

CHEMICAL METHODS

Used by methods of fixation in processing of tissue for

histopathological diagnosis

Utilizes organic or non-organic solutions to maintain

adequate morphological preservation

3 major categories

o Coagulant Fixatives

o Compound Fixatives

o Cross-link Fixatives

A.COAGULANT FIXATVES

Both organic and inorganic solutions may

coagulate proteins making them insoluble

Results in cytoplasmic flocculation as well as

poor preservation of mitochondria and

secretory granules

Not useful in ultrastructural analysis

TYPES OF COAGULANT FIXATIVES

1.DEHYDRANT COAGULANT FIXATIVESo Most commonly used are ALCOHOL and

ACETONE

oAlcohol denatures protein differently

depending on:

Choice and concentration of

alcohol

Presence of organic and

inorganic substances

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pH

temperature

o METHANOL has a closer structure with water

> Fixation begins at 80% conc. or more

o

ETHANOL 00 competes more strongly in interactionwit hhydrophobic areas of molecues

> Begins at a conc. of 50% - 60%

ACIDIC COAGULANTS change and charges on the

ionisable side chains of the proteins and disrupt

electrostatic and hydrogen bonding

oACETIC ACID coagulates nucleic acids but

does not fix or precipitate proteins added to

other fixatives to prevent loss of nucleic acids

o PICRICA ACID or TRINITROPHENOL Slightly dissolves in water to form a

weak acid solution

Produces brighter staining

May cause hydrolysis and loss of

nucleic acids due to low pH

B.CROSS-LINK FIXATIVES

Has potential actions forming cross-links

between proteins and nucleic acids or between

nucleic acids and proteins

A.k.a. COVALENT ADDITIVE FIXATIVES

Examples:

o Formaldehyde

o Chloral hydrate

o Glutaraldehyde

o Glyoxal

C.COMPOUND FIXATIVES

Useful for specific tissues

E.g. ALCOHOLIC FORMALIN for fixation of

FATTY TISSUE

LIPID FIXATION

Lipids are largely removed during preparation if tissue.

Cryostat or frozen section should be used fordemonstrating lipid in tissue.

Fixatives containing mercuric chloride or potassium

dichromate can be effective for preservation of lipids in

cryostat.

Phospholipids are fixed by aldehydes Bakers formol-

calcium.

Ultrastructural fixation of lipids is achieved by post-

fixing inimidazole osmium tetroxide

Cholesterol may by fixed by digitonin for

ultrastructural demonstration.

PROTEIN FIXATION

Neutral buffered formol saline orformaldehyde

vapour most commonly used fixative foramino acid

histochemistry.

CARBOHYDRATE AND GLYCOGEN FIXATION

Alcoholicfixatives are generally recommended for

glycogenfixation.

Alcoholic formaldehydeis better fixative for humanskinthan neutral buffered formaldehye.

Rossmans fluidandcold absolute alcohol most

useful fixative for preservingglycogen.

Celloidin coating for better retention of glycogen.

MIXTURE FIXATIVES

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KARNOVSKY PARAFORMALDEHYDE-

GLUTARALDEHYDE two glutaraldehyde fixative

mixtures useful forelectron cytochemistry.

ACROLEIN introduced as a mixture with

formaldehyde and glutaraldeydeo Penetrates tissue rapidly preserving

morphology and enzyme activity at low

concentrations.

o Useful for immersion fixations forsurgical

biopsies.

ALDEHYDE FIXATIVES

Formaldehyde

10% Formol-saline

10% Neutral buffered formalin Formal-corrosive

Alcoholic formalin

Glutaradehyde

FORMALDEHYDE

FORMALIN

Most widely used fixative

A gas produced by the oxidation of methyl alcohol

A saturated solution of formaldehyde gas in water

approximately 35-40% gas by weight 10% FORMALIN a solution 10 ml formalin with 90

ml water

FIXATION TIME: 24 hours

Usually buffed to pH 7 with phosphate buffer

High formaldehyde concentrates tend to over-harden

the outer layer of the tissue and adversely affect the

staining.

PRECAUTIONS

o Room should be properly ventilated with

adequate windows and exhaust fan to prevent

inhalation of the fumes and consequent injury

the eyes and the nose.

o Dermatitis can be avoided by the use of rubbergloves.

oAcid reaction due to formic acid formation can

be buffered or neutralized by adding

MAGNESIUM CARBONATE or CALCIUM

CARBONATE to 10-15% formalin.

o In TISSUE CONTAINING MUCH BLOOD

unbuffered formalin leads to the formation of

dark brown pigment granules.

Removal of formalin pigment can be achieved by eitherof the following:

KARDASEWITSCHs METHOD

70% ethyl alcohol + 28% ammonia

water for 5 mins to 3 hours

o LILIES MATHOD

Acetone + 3 vol of hydrogen peroxide +

28% ammonia water for 1 to 5 mins

followed by 70% + running water

o PICRIC ACID

For five minutes to 2 hours and the

wash in running tap water for 10 15

minutes

10% FORMOL SALINE

A simple microanatomical fixative made up of

saturated formaldehyde(40% by weight volume)

deluted with 10% SODIUM CHLORIDE

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Recommended for fixation ofcentral nervous tissues

andgeneral post-mortem tissuesforhistochemical

examination.

FORMULATION:

o Formaldehyde 40 mlo NaCl 9 mg

o Distilled water 900 ml

FIXATION TIME

o 24 hours at 35C

o 48 hours at 20-25C

10% NEUTRAL BUFFERED FORMALIN

PHOSPHATE BUFFERED FORMALIN (7pH)

Recommended for preservation ofstorage surgical,

post-mortemandresearch specimen.

FORMULATION

o Sodium dihydrogen phosphate

o Disodium hydrogen phosphate

o Formaldehyde

o Distilled water

FIXATION TIME: 4 -24 hours

Best fixative fortissue containing iron pigmentand

forelastic fiberwhich do not stain well after Susa,

Zenker, and Chromatic fixation.

Require NO post treatment after fixationFORMAL-CORROSIVE

FORMAL-SUBLIMATE

FORMAL MERCURIC CHLORIDE solution is

recommended for fixation of routinepost-mortem

tissue.

FORMULATION:

o Sat aq. Mercuric chloride 90 ml

o Formaldehyde 10 ml

FIXATION TIME: 3 24 hours

Penetrates small pieces of tissue rapidly

There is no need for washing out can be transferred

directly from fixative to alcohol.

It forms mercuric chloride deposits. It inhibits the determination of the extent of tissue

decalcification.

ALCOHOLIC FORMALIN

GENRDES Fixative

Can be used forrapid diagnosis it fixes and

dehydrates at the same time

Good for preservation of glycogen and for

microincineration technique

FORMULATION:

o 95% ethyl alcohol sat.

with picric acid 80 ml

o Strong formaldehyde

solution 15 ml

o Glacial acetic acid 5 ml

GLUTARALDEHYDE

Made up of two formaldehyde residues linked up by

three carbon chains.

Used for electron microscopy buffered

glutaraldehydethenosmium tetroxide 2.5% solution used forsmall tissue fragmentsand

tissue biopsiesfixed in 2-4 hours at room temp.)

4% solution recommended for large tissue

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Specimen vial must be kept refrigerated during fixation

process.

Solution may be changed several times during fixation

by swirling the vials.

FIXATIVE FIXATION TIMEFormaldehyde 24 hours

Formol-saline 24 hours (35C)

48 hours (20-25C)

10% Neutral buffered

Formalin 4-24 hours

Formal-corrosive 3-24 hours

Alcoholic formalin rapid

Glutaraldehyde

- 2.5% 2-4 hours @ RT

- 4% 6-8 to 24 hours

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