Types of Fixative

I.

8.

According to COMPOSITION
A. SIMPLE FIXATIVES are made up of only one
component substance
1. Aldehydes
a. 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

II.

constituents.
According to ACTION
A. MICROANATOMICAL FIXATIVES those that
permit the general microscopic study of tissue
structures

without

altering

the

structural

pattern 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

Zenker-formol

B. CYTOLOGICAL
preserve

FIXATIVES

specific

parts

those

and

that

particular

microscopic elements of the cell itself

3 Types of Cytological Fixatives
o
o
o

Nuclear fixative
Cytoplasmic fixative
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

3. HISTOCHEMICAL FIXATIVES those that preserve the

CHEMICAL constituents of the cells and tissues.
Formol Saline 10%
Absolute Ethyl Alcohol
Newcomers fluid

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 a
vacuum chamber at -40C.

Fixation of tissue can be accomplished by:

CHEMICAL METHODS

a. PHYSICAL METHOD
Heating
Freeze-drying
Microwaving
b. CHEMICAL METHOD
Coagulant fixatives
Compound fixatives
Cross-linking fixatives
PHYSICAL 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.

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 FIXATIVES
o Most commonly used are ALCOHOL and
o

ACETONE
Alcohol denatures protein differently
depending on:
Choice

alcohol
Presence

and
of

concentration
organic

of
and

inorganic substances

o
o

pH
temperature
METHANOL has a closer structure with water
> Fixation begins at 80% conc. or more
ETHANOL 00 competes more strongly in interaction
wit hhydrophobic areas of molecues
> Begins at a conc. of 50% - 60%

FATTY TISSUE
LIPID FIXATION

Lipids are largely removed during preparation if tissue.

Cryostat or frozen section should be used for

demonstrating lipid in tissue.

Fixatives containing mercuric chloride or potassium
dichromate can be effective for preservation of lipids in

ACIDIC COAGULANTS change and charges on the

ionisable side chains of the proteins and disrupt
electrostatic and hydrogen bonding
o ACETIC ACID coagulates nucleic acids but
does not fix or precipitate proteins added to
o

other fixatives to prevent loss of nucleic acids

PICRICA ACID or TRINITROPHENOL
Slightly dissolves in water to form a

weak acid solution

Produces brighter staining
May cause hydrolysis and

cryostat.
Phospholipids are fixed by aldehydes Bakers formol-

calcium.
Ultrastructural fixation of lipids is achieved by post-

fixing in imidazole osmium tetroxide

Cholesterol
may
by
fixed
by
digitonin

PROTEIN FIXATION
Neutral buffered formol saline or formaldehyde
vapour most commonly used fixative for amino acid

of

nucleic acids due to low pH

B. CROSS-LINK FIXATIVES
Has potential actions

for

ultrastructural demonstration.

loss

E.g. ALCOHOLIC FORMALIN for fixation of

histochemistry.
CARBOHYDRATE AND GLYCOGEN FIXATION

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

Alcoholic fixatives are generally recommended for

glycogen fixation.
Alcoholic formaldehyde is better fixative for human

skin than neutral buffered formaldehye.

Rossmans fluid and cold absolute alcohol most

useful fixative for preserving glycogen.

Celloidin coating for better retention of glycogen.

MIXTURE FIXATIVES

KARNOVSKY
GLUTARALDEHYDE

PARAFORMALDEHYDE

two

glutaraldehyde

fixative

mixtures useful for electron cytochemistry.

ACROLEIN introduced as a mixture
formaldehyde and glutaraldeyde
o Penetrates
tissue
rapidly
morphology
o

and

enzyme

at

with

low

ventilated

with

the eyes and the nose.

Dermatitis can be avoided by the use of rubber

gloves.
Acid reaction due to formic acid formation can
be

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

neutralized

CARBONATE

by

or

adding

CALCIUM

CARBONATE to 10-15% formalin.

In TISSUE CONTAINING MUCH BLOOD
dark brown pigment granules.

Removal of formalin pigment can be achieved by either

of the following:

FORMALDEHYDE
FORMALIN
Most widely used fixative
A gas produced by the oxidation of methyl alcohol
A saturated solution of formaldehyde gas in water

or

unbuffered formalin leads to the formation of

Formaldehyde
10% Formol-saline
10% Neutral buffered formalin
Formal-corrosive
Alcoholic formalin
Glutaradehyde

buffered

MAGNESIUM

ALDEHYDE FIXATIVES

properly

concentrations.
Useful for immersion fixations for surgical

staining.
PRECAUTIONS

be

inhalation of the fumes and consequent injury

biopsies.

should

adequate windows and exhaust fan to prevent

preserving

activity

Room

KARDASEWITSCHs METHOD
70% ethyl alcohol + 28% ammonia
water for 5 mins to 3 hours
LILIES MATHOD
Acetone + 3 vol of hydrogen peroxide +
28% ammonia water for 1 to 5 mins

followed by 70% + running water

PICRIC ACID
For five minutes to 2 hours and the
wash in running tap water for 10 15
minutes

10% FORMOL SALINE

simple

microanatomical

fixative

made

up

of

saturated formaldehyde (40% by weight volume)

deluted with 10% SODIUM CHLORIDE

Recommended for fixation of central nervous tissues

and general post-mortem tissues for histochemical

examination.
FORMULATION:
o Formaldehyde
40 ml
o NaCl
9 mg
o Distilled water
900 ml
FIXATION TIME
o 24 hours at 35C
o 48 hours at 20-25C

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 for rapid diagnosis it fixes and

PHOSPHATE BUFFERED FORMALIN (7pH)

Recommended for preservation of storage surgical,

dehydrates at the same time

Good for preservation of

post-mortem and research specimen.

FORMULATION
o Sodium dihydrogen phosphate
o Disodium hydrogen phosphate
o Formaldehyde
o Distilled water
FIXATION TIME: 4 -24 hours
Best fixative for tissue containing iron pigment and

10% NEUTRAL BUFFERED FORMALIN

FORMAL-CORROSIVE

FORMAL-SUBLIMATE
FORMAL
MERCURIC

CHLORIDE

solution

is

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

Made up of two formaldehyde residues linked up by

three carbon chains.

Used
for
electron

glutaraldehyde then osmium tetroxide

2.5% solution used for small tissue fragments and

tissue biopsies fixed in 2-4 hours at room temp.)

4% solution recommended for large tissue <4 mm

thick (fixed in 6-8 hours up to 24 hours)

FIXATION TIME: hours to 2 hours
Preserves cellular structure better recommended for

recommended for fixation of routine post-mortem

tissue.
FORMULATION:
o Sat aq. Mercuric chloride 90 ml
o Formaldehyde
10 ml

and

GLUTARALDEHYDE

for elastic fiber which do not stain well after Susa,

Zenker, and Chromatic fixation.
Require NO post treatment after fixation

glycogen

microscopy

buffered

enzyme histochemistry and electron microscopy

Specimen vial must be kept refrigerated during fixation

10% Neutral buffered

process.
Solution may be changed several times during fixation

Formalin

4-24 hours

Formal-corrosive

3-24 hours

by swirling the vials.

Alcoholic formalin

rapid

FIXATIVE

FIXATION TIME

Formaldehyde

24 hours

Formol-saline

24 hours (35C)

Glutaraldehyde
-

2.5%
4%

2-4 hours @ RT
6-8 to 24 hours

48 hours (20-25C)