FIXATION

 First and most critical step in histotechnology that involves fixing or preserving fresh
tissue for examination
 Primary Aim:
o Preserve the morphologic and chemical integrity of the cell in a life-like manner
as possible
 Secondary Goal
o Harden and protect the tissue from the trauma of further handling, so that it is
easier to cut during gross examination

FIXATIVE ADVANTAGES DISADVANTAGES NOTES

ALDEHYDE FIXATIVES
Formaldehyde •Cheap, readily available, •Soft fixative ✓10%- most widely
easy to prepare used
✓ produced by the
oxidation of methyl
alcohol

✓ Soluble in water to
the extent of 37-40%
weight in

volume

✓ High
concentrations tend
to over-harden the
outer

layer of the tissue

and affect staining
adversely

✓ FT: 24 hours

10% Formal •Easy sectioning •Slow fixative ✓made up of

Saline •Good staining saturated
•less shrinkage formaldehyde (40%,
•even fixation by weight

volume) diluted to
10% with sodium
chloride

✓ For CNS and

general post-mortem
tissues
 for histochemical
examination

✓ FT: 12-24 hrs (24

hrs at 38oC ; 48 hrs
at 20-25oC)
10% Neutral •Most routine purpose •Lost of basophilic ✓Most commonly
Buffered •Fixed tissue rapidly staining of the used and best
Formalin •Stops formation of cytoplasm and general tissue
formalin pigment nucleus
• fixative in histology

✓ For surgical, post

mortem, research
specimens

✓ Usually buffered
with phosphate buffer
(pH of 7.2-

7.4)

✓ FT: 4-24 hrs

Formol- • Rapid fixation with little •Leaves deposit of
Corrosive shrinkage mercuric chloride in ✓ Fixes lipids,
(Formol •Recommended for routine the tissue especially neutral fats
sublimate) post-mortem tissues •Will not allow and phospholipids
cutting by frozen
sections ✓ FT: 3-24 hours

Karnovsky’s •Suitable for use when

Fixative preparing samples for light ✓ mixture of
microscopy in resin paraformaldehyde
embedding and sectioning, and glutaraldehyde
and for electron
microscopy ✓ for electron
cytochemistry
Glutaraldehyde •Penetration rate slightly •Can cause
faster shrinkage of cells ✓ causes rapid and
•Enzymatic activity partially at higher irreversible changes,
preserved which permits concentration fixes quickly
cytochemical localization
following fixation ✓ For enzyme
histochemistry and
EM studies

✓ Should be stored
at 4oC and pH 5

✓ 2.5%: 2-4 hrs at

 RT (for small tissue
fragments

and needle biopsies)

✓ 4%: 6-8 hours up

to 24 hours (larger

tissues)
Acrolein •Causes little shrinkage •Highly toxic
•Reacts rapidly with •Highly flammable ✓ is another aldehyde
proteins and extremely which has been
•Good for fixation of large reactive (explosive) introduced as a
blocks of tissues •Destroys most
enzymatic activities mixture with
•Solubilizes lipids glutaraldehyde or
formaldehyde

✓ It penetrates
tissues rapidly,
preserves
morphology

and enzyme activity

at low concentrations,
and may

be used for
immersion fixation of
surgical biopsies
ALCOHOLIC FIXATIVES
Methanol 100% •Fixes and dehydrates at •Slow penetration
the same time ✓ For blood smears
•Excellent for fixing dry and bone marrows
and wet smears and bone
marrow tissues
Ethanol (Ethyl •Fixes blood tissue films •Inadequately ✓ Both as a fixative
alcohol) and smears preserve and dehydrating
•Fixes tissue pigments leukocytes agent
fairly well •It is a strong
•It is ideal for small tissue reducing agent ✓ most commonly
fragments •Tissue left in used for exfoliative
alcohol too long will study
shrink

✓ FT: 18-24 hrs

Isopropanol 95% •It is used for fixing touch
preparations ✓ For fixing touch
preparations
Carnoy’s •Excellent nuclear fixation •Destroys RBC
Fixative •Penetrates rapidly ✓ Most rapid fixative

✓ both fixative and

dehydrating agent

✓ Fixes brain tissue

and diagnosis of
Rabies

✓ FT: 1-3 hrs(6 hrsfor

brain)

Clarke’s •Good penetration

Solution •Nuclear details ✓ used on frozen
cytoplasmic elements sections and smears

✓ FT: 3-4 hrs

Alcoholic •Sometimes used during

Formalin processing complete ✓ can be used for
fixation following fixation or post-
incomplete primary fixation of large fatty
formalin fixation
specimens

✓ FT: 12-24 hrs

Formol-Acetic •Sometimes used to fix
Alcohol diagnostic cryostat ✓ sometimes used to
sections. fix diagnostic cryostat
•Faster acting agent than sections
alcoholic formalin
✓ FT: 1-6 hrs

Gendre’s •Fixation is faster. •Produces griss

Fixative •It is used to fix sputum, hardening of tissue ✓ used to fix sputum,
(Alcoholic since it coagulates mucus •Causes partial since it coagulates
Bouins) lysis of RBC mucus

✓ cause better
retention of
carbohydrate
(glycogen) in

tissues

✓ FT: between 4 hrs

and overnight
followed by washing
 in 70% ethanol
followed by 95%
ethanol
Newcomer’s • Recommended for fixing
Fluid MPS(MucoPolysaccharide) ✓ Better reaction in
and nuclear proteins Feulgen stain than
Carnoy's fluid

✓ Both as a nuclear
and histochemical
fixative

✓ FT: 12-18 hrs at

3oC
METALLIC FIXATIVES
Mercuric permits brilliant Penetrates poorly
Chloride metachromatic staining of and causes tissue ✓ MOST COMMON
cells shrinkage so it is metallic fixative
usually combined
with other fixatives ✓ the routine fixative
of choice for
preservation of cell

detail in tissue
photography
Zenker’s •It produces a fairly rapid •Pemetration is
Solution (with and even fixation of tissues slow ✓ For blood
GAA) •Recommended for (congested)
trichrome staining specimens

✓ For trichrome
staining

✓ For fixing small

pieces of liver,
spleen, CT fibers and

nuclei

✓ FT: 12-24 hrs

Zenker’s Formol •Pemetrates and fixes

(Helly’s tissue well ✓ fixative for pituitary
Solution) gland, BM and
extramedullary
hematopoiesis and
intercalated discs

✓ FT: 12-24 hrs

Heidenhain’s •Minimum shrinkage •Tissue hardening
Susa Solution •Brilliant staining results ✓ Recommended
•Rapid fixation mainly for tumor
biopsies

especially of the skin;

it is an excellent
cytologic

fixative

✓ FT: 3-12 hrs

B5 (Lillies) •Important for identifying

Fixative normal and abnormal cells. ✓ important for
identifying normal
and abnormal cell

types in bone marrow

(hematopoietic
tissue)

specimens

✓ FT: 4-8 hrs

OXIDIZING AGENTS
Osmium •Acts as electron stain as •Slow rate of
Tetroxide well as fixative penetration ✓ traditionally used in
•Good preservation of •Soluble in certain electron microscopy
cellular structures lipids both as a
•Only partially
preserve proteins fixative and a heavy
metal stain

✓ excellent stain for

lipids in membranous
structures

and vesicles

✓ should be kept in
DARK COLORED,
chemically clean

bottle to prevent
evaporation and
reduction by

sunlight and organic

matter
✓ produces BLACK
 PRECIPITATE
(Osmium Oxide)

✓ Prevention:

✓ Remedy
Flemming’s •Permanently fixes fats •It is a poor
Solution penetrating agent ✓ the most common
chrome-osmium
acetic acid

fixative used for

nuclear preparations

✓ FT: 24-48 hrs

Flemming’s
Solution without ✓ made up only of
Acetic Acid chromic and osmic
acid

✓ recommended for
cytoplasmic
structures

particularly the
mitochondria

✓ removal of acetic
acid from the formula
serves to

improve the
cytoplasmic detail of
the cell

✓ FT: 24-48 hrs

CHROMATE FIXATIVES
Chromic Acid •Preserves carbohydrates
✓ used in 1-2%
aqueous solution

✓ preserves
carbohydrates

✓ a strong oxidizing
agent hence strong
reducing agent

must be added
Potassium •It preserves mitochondria
Dichromate ✓ used in 3%
aqueous solution

✓ preserves
mitochondria at pH
4.5-5.2

Regaud’s •It penetratres tissue well.

(Mueller's) Fluid ✓ for the
demonstration of
chromatin,
mitochondria, golgi
bodies and RBC

✓ FT: 12-48 hrs

Orth’s Fluid • It is recommended for
study of degenerative ✓ For study of early
processes and tissue degenerative
necrosis processes and tissue

necrosis

✓ Demonstrates
Rickettsia and other
bacteria

✓ FT: 36-72 hrs

PICRIC ACID FIXATIVES
Bouin’s Solution •Rapid penetration •Lyses red cells ✓ recommended for
fixation of Embryo's
and Pituitary glands.

✓ stains tissue bright

yellow due to picric
acid

✓ Excellent fixative
for glycogen
demonstration

✓ FT: 4-18 hrs;

stored at RT
Hollande’s
Solution
✓ recommended for
gastro-intestinal tract
specimens

and fixation of
 endocrine tissues

✓ FT: 4-18 hrs

Brasil's
Alcoholic
Picroformol
Fixative
OTHER FIXATIVES
Lead Fixatives

✓ used in 4%
aqueous solution of
basic lead acetate

✓ Stable at alkaline
pH

✓ recommended for
acid
mucopolysaccharides

Trichloroacetic )
Acid (TCA
✓ Precipitates
proteins

✓ Poor penetrating
agent, hence, is
suitable only for

small pieces of
tissues or bones

PHYSICAL METHODS OF FIXATION

Heat Fixation

✓ Involves thermal
coagulation of tissue
proteins for rapid

diagnosis

✓ For frozen tissue

sections and
preparation of
bacteriologic

smears
 ✓ Primarily used to
accelerate other
forms of fixation

Microwave
Fixation
✓ the tissue is heated
right through the
block in a very short

time, thereby
potentially allowing
the study of cellular

processes that
proceed very rapidly

✓ Glyoxal-based
fixatives heated at
55oC: efficient
method of

microwave fixation