FIXATION

Medical Laboratory Science Program

College of Allied Medical Professions
Lyceum of the Philippines University-Batangas
FIXATION
 First and most critical step in histotechnology.
 Process that preserves tissues from decay, thereby
preventing autolysis and putrefaction.
 The process should be carried out as soon as possible
after removal of tissue from the body.
 Goals of fixation:
1. Preserve the morphologic and chemical integrity
of the cell in as life – like manner as possible.
(PRIMARY AIM) 
2. Harden and protect the tissue from the trauma of
further handling.
FIXATION

Objectives of fixation:
 To preserve the tissue (stops all cellular
activities)
 To prevent breakdown of cellular elements
(prevent autolysis and putrefaction).
 To coagulate or precipitate protoplasmic
substances (renders tissue components
insoluble)
FIXATION
Methods of fixation:
 Physical means – heating, microwave
fixation and cryo – preservation.
 Chemical means:
 Immersion fixation (tissues are immersed
in fixatives)
 Perfusion fixation (tissues are perfused or
injected with fixatives)
FIXATION

Two Basic Mechanisms Involved in Fixation:

 Additive fixation – a process whereby the
fixative is taken in by the tissue and becomes
part of the tissue by forming cross-links or
molecular complexes giving stability to
proteins.
 Examples are formalin, mercury and
osmium tetroxide.
FIXATION

Two Basic Mechanisms Involved in Fixation:

 Non-Additive Fixation – a process whereby the
fixing agent is not incorporated in the tissue but
alters the tissue composition and stabilizes the
tissue by removing water. If water is removed,
new cross-links are established within the
protein molecules.
 Examples are alcoholic fixatives.
FIXATION

So far, no single fixative has yet been

known to possess all the qualities of an ideal
solution. There are numerous fixatives
available, and each has its own advantages
and disadvantages.
FIXATION

Benefits of fixation:
 Allows thin sectioning of tissue by
hardening tissues
 Prevents autolysis and inactivates infectious
agents (except prion diseases).
 Improves cell avidity for special stains.
FIXATION
Practical considerations to optimize fixation of tissues:
 Transfer specimens to fixative quickly (less than 1
hour)
 Provide sufficient volume of fixatives (fixative to
tissue ration of 20:1 or at least 10:1)
 Remove anatomical barriers (fascia, bone, feces, thick
tissue, etc.)
 Large specimens must be sectioned or inflated with
fixatives (lungs) or opened and cleaned (GI
specimens)
FIXATION
Practical considerations to optimize fixation of tissues:
 Contaminated fixatives with bile, blood, feces and
others must be replaced to ensure effectiveness.
 Pinning specimens to a corkboard or inserting a paper
or gauze “wick” into tubular structures can improve
fixation and reduce tissue distortion.
 Allow sufficient fixation time; rates of penetration vary
according to fixative types.
 Allow adequate permeation of fixatives through the
cassettes.
FIXATION

Practical considerations to optimize fixation of

tissues:
 Prolonged fixation may be more difficult to
reverse and may result in loss of
immunohistochemical antigenicity.
 The tissue hardens upon fixation and remains in
whatever shape it was fixed in. This should be
considered in cases where the final orientation of
the sections is important.
FIXATION
Main factors involved in fixation:
 Volume – Fixatives should be 10 – 20 times the
volume of the tissue to be fixed. the most common
error in histotechnology is insufficient fixative to
tissue ratio. Change the fixative solutions at intervals
to avoid exhaustion of the fixative.
 Agitation – enhance fixation of the specimen.
 Hydrogen ion concentration (pH) – fixation is best
carried out close to neutral pH, in the range of 6 – 8.
FIXATION
Main factors involved in fixation:
 Temperature – fixation employing automation
(Autotechnicon) usually work at 40oC. Generally,
fixation at room temperature is sufficient to
maintain excellent morphological detail.
NOTE: Increasing the temperature will increase the
speed of fixation, however, will also increase the rate
of autolysis and will actually “cook” the tissue. Cold
temperatures retard fixation.
FIXATION
Main factors involved in fixation:
 Thickness of sections – Tissue blocks for electron
microscopy should only be (1 – 2 mm2) and 2 cm2
and no more than 4 mm thick for light
microscopy. This should not be compromised in
order to obtain full penetration and satisfactory
fixation.
NOTE (update): Aldehyde fixatives generally has a
penetration rate of 2 to 3 mm/hour. (Old book –
formalin penetration rate is 1 mm/hour).
FIXATION
Main factors involved in fixation:
 Osmolality
 Hypertonic fixatives give rise to cell
shrinkage.
 Hypotonic fixatives cause swelling and poor
fixation.
 The best results are actually obtained using
a slightly hypertonic solution (400-450
mOsm) Isotonic solutions are at 340 mOsm.
FIXATION

Main factors involved in fixation:

 Concentration of Fixatives
 Formaldehyde is usually used as a 10%
solution
 Glutaraldehyde is used as 3% solution.
 These concentrations are found to be an
ideal factor for effective fixation of tissues.
FIXATION

Main factors involved in fixation:

 Duration of fixation – some tissues take longer
to fix than others depending on their structure.
Fibrous organs such as uterus or intestinal
tract take longer to be fixed than loosely
textured tissues from biopsy or scrapings.
 Fixation time can be cut down by using heat,
vacuum, agitation or microwave.
FIXATION

Main factors involved in fixation:

 Duration of fixation – prolonged fixation may
cause shrinkage and excessive hardening of
tissues. Incomplete fixation causes softening of
tissues making the tissue very hard to cut
during section-cutting.
 Time interval – samples should be fixed
immediately after removal or death.
FIXATION
Effects of fixatives in general:
 They reduce the risk of infections during handling
and actual processing of tissues.
 They harden soft and friable tissues and make the
handling and cutting of sections easier.
 They make the cells resistant to damage and
distortion caused by hypotonic and hypertonic
solutions used in tissue processing.
FIXATION

Effects of fixatives in general:

 They inhibit bacterial decomposition.
 They increase the optical differentiation of
cells and tissue components rendering them
more readily visible during examination.
 They act as mordants or accentuators to
promote and hasten staining.
 FIXATION
Characteristics of a good fixative:
 It must be cheap.
 It must be stable.
 It must be safe to handle.
 It must kill the cell quickly thereby producing
minimum distortion of cell constituents.
 It must inhibit bacterial decomposition and
autolysis.
 It must produce minimum shrinkage of tissue.
FIXATION
Characteristics of a good fixative:
 It must permit rapid and even penetration of
tissues.
 It must harden tissues thereby making the
cutting of sections easier.
 It must be isotonic causing physical and
chemical alteration of cells (although this is not
a strict rule)
FIXATION

Characteristics of a good fixative:

 It must make cellular components insoluble to
hypotonic solutions and render them
insensitive to subsequent processing.
 It must permit the subsequent application of
many staining procedures to facilitate easier
and more profitable examination.
FIXATION
Types of fixatives:
 According to Composition
 Simple fixatives – are made up of only one
component substance.
 Compound fixatives – are those that are
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 and tissue
components.
FIXATION
SIMPLE
FIXATIVES Picric acid
Aldehydes
Acetic acid
Formaldehyde
Glutaraldehyde Acetone

Metallic Fixative Alcohol

Mercuric chloride Osmium
Chromate fixatives tetroxide
FIXATION
Types of fixatives:
 According to Action
 Microanatomical fixatives - are those that
permit the general microscopic study of tissue
structures without altering the structural
pattern and normal intercellular relationship
of the tissues in question.
 Cytological fixatives – those that preserve
specific parts and particular elements of the
cell itself.
FIXATION
MICROANATOMICA
L FIXATIVES
10% formol-saline Zenker’s solution
10% neutral buffered Zenker-formol
formalin (Kelly’s solution)
Heidenhain’s SuSa Bouin’s solution
Formol sublimate Brasil’s solution
(Formol corrosive)
FIXATION
Types of fixatives:
 According to Action
 Cytological Fixatives
 Nuclear fixatives – preserve nuclear structures.
Contains glacial acetic acid as a primary
component (pH < 4.6).
 Cytoplasmic fixatives – preserves cytoplasmic
structures. They should NOT contain glacial
acetic acid which destroys mitochondria and
Golgi bodies (pH >4.6).
FIXATION
NUCLEAR CYTOPLASMIC
FIXATIVES FIXATIVES
Flemming’s fluid Flemming’s fluid without
Carnoy’s fluid acetic acid
Kelly’s fluid
Bouin’s fluid
Formalin with post-
Newcomer’s fluid
chroming
Heidenhain’s SuSa
Regaud’s fluid (Muller’s
fluid)
Orth’s fluid
FIXATION

Types of fixatives:
 According to Action
 Cytological Fixatives
 Histochemical fixatives – those that
preserves chemical constituents of cells and
tissues.
FIXATION

HISTOCHEMICAL FIXATIVES
10% formol-saline
Absolute ethyl alcohol
Acetone
Newcomer’s fluid
FIXATION
Secondary fixation
 It is the process of placing an already fixed tissue in a
second fixative in order to:
 facilitate and improve demonstration of particular
substances.
 to make special staining techniques possible by
acting as a mordant.
 to ensure further and complete hardening and
preservation of tissues.
 This process is usually done before dehydration and
on deparaffinized sections before staining.
FIXATION
Post chromatization
 A form of secondary fixation where a primarily fixed
tissue is placed in an aqueous solution of 2.5 to 3%
potassium dichromate to act as a mordant.
Washing out
 It is the process of removing excess fixative from the
tissue after fixation in order to improve staining and
remove artifacts from tissues.
 Examples are tap water, 50 – 70% alcohol and
alcoholic iodine.
CHEMICAL FIXATIVES
CHEMICAL FIXATIVES
Two major groups of chemical fixatives:
 Crosslinking fixatives (aldehydes) – fixatives that
act by creating covalent chemical bonds between
proteins in tissue.
 Precipitating (or denaturing) fixatives (alcoholic
fixatives) – fixatives that act by reducing the
solubility of protein molecules by disrupting
hydrophobic interactions stabilizing protein
molecules.
ALDEHYDE FIXATIVES
 Formaldehyde is a gas and formalin is a liquid.
 Formaldehyde (37% w/v) = 37 grams of
formaldehyde in 100 mL of solution.
 Formaldehyde (37%) + water = formalin (unstable)
 form paraformaldehyde (turbid)
 Stabilizing agent: 10 – 15% methanol
 Chemical composition of concentrated formalin: 37 –
38% of formaldehyde, 10 – 15% of methanol and 48 –
53% water. Therefore, a fixative labeled as 10%
buffered formalin is actually only a 4% solution of
formaldehyde.
ALDEHYDE FIXATIVES
Advantages of using formalin:
 Cheap, readily available, easy to prepare and
relatively stable if stored in buffered solution.
 Compatible with many stains.
 Does not over harden tissues even with prolonged
periods as long as solutions are regularly changed.
 Penetrates tissues well.
 Preserves fats, mucins, glycogen.
 Preserves but does not precipitate proteins (enzyme
study).
 Recommended for colored tissue photography.
 Does not require washing out.
ALDEHYDE FIXATIVES
Disadvantages of using formalin:
 Fumes are irritating to the eyes and nose. The liquid
may cause skin irritation. May cause lung and
nasopharyngeal CA.
 May produce considerable shrinkage of tissues.
 Soft fixative and does not harden some cytoplasmic
structures.
 If unbuffered (formic acid will accumulate):
 Formalin reduces both basophilic and eosinophilic
staining of cells.
 It causes brown pigment granules on blood
containing organs (due to hemoglobin).
ALDEHYDE FIXATIVES

Disadvantages of using formalin:

 If unbuffered (continuation):
 The brown pigment maybe mistaken as malarial
parasites and melanin pigments. REMEDY  add
MgCO3 or CaCO3 as buffer.

REMOVAL OF FORMALIN PIGMENTS:

 Kardasewitch- (ammonia water + ethyl alcohol)
 Lillie’s Method- (ammonia water + alcohol + acetone
+ hydrogen peroxide)
 Picric acid
ALDEHYDE FIXATIVES

Disadvantages of using formalin:

 Prolonged fixation may produce:
 Bleaching of the specimen and loss of natural
tissue colors.
 Dispersal of fat from the tissue into the fluid
 Dissolution or loss of glycogen and urate
crystals.
ALDEHYDE FIXATIVES
10% Formal – Saline (pH 6.8)
 Fixation time: 12 – 24 hours
 Made up of saturated formaldehyde (37 – 40%)
diluted to 10% with sodium chloride (HCHO +
NSS).
 It is recommended for fixation of CNS (brain)
tissues and general post mortem tissues for
histochemical examination.
 It is also recommended for the preservation of
lipids, especially phospholipids.
ALDEHYDE FIXATIVES

10% Formal – Saline (pH 6.8)

Advantages:
 It penetrates and fixes tissues evenly.
 It is ideal for most staining techniques, including
silver impregnation.
 It allows natural tissue color to be restored upon
immersion in 70% alcohol.
ALDEHYDE FIXATIVES

10% Formal – Saline (pH 6.8)

Disadvantages:
 Slow fixative.
 Formal – saline fixed 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.
ALDEHYDE FIXATIVES

10% Neutral Buffered Formalin (NBF) (pH 7)

 HCHO+NaH2PO4 + Na2H2PO4
 Fixation time: 24 hours at RT
 Most widely used fixative for routine histology.
 Fixative of choice for procedures requiring
paraffin embedding.
ALDEHYDE FIXATIVES
10% Neutral Buffered Formalin (NBF) (pH 7)
Advantages (similar to 10% formal saline), and:
 It prevents precipitation of acid formalin
pigments on post – mortem tissue.
 It is the best fixative for tissues containing iron
pigments and for elastic fibers which do not stain
well after Susa, Zenker and chromate fixation.
 It requires no post – treatment after fixation and
goes directly into 80% alcohol for processing.
ALDEHYDE FIXATIVES

10% Neutral Buffered Formalin (NBF) (pH 7)

Disadvantages:
 It is longer to prepare; hence, time consuming.
 Positivity of mucin to PAS is reduced.
 Reactivity of Myelin to Weigert’s iron
hematoxylin stain is reduced.
 It is inert (chemically inactive) towards lipids,
especially neutral fats and phospholipids.
ALDEHYDE FIXATIVES

Zinc formalin (unbuffered)

 Alternative to mercuric chloride fixatives
(metallic fixatives).
ALDEHYDE FIXATIVES

Formol – Corrosive (Formol – Sublimate)

 HCHO + Mercuric Chloride
 Fixation time: 3 – 24 hours
 Recommended for routine post mortem
examination.
ALDEHYDE FIXATIVES
Formol – Corrosive (Formol – Sublimate)
Advantages:
 It penetrates small pieces of tissues rapidly.
 It is excellent for many staining procedures
including silver reticulum methods.
 Cytological structures and blood cells are well –
preserved. There is no need for “washing out”.
Tissues can be transferred directly from fixative
to alcohol.
 It fixes lipids, especially neutral fats and
phospholipids.
ALDEHYDE FIXATIVES

Formol – Corrosive (Formol – Sublimate)

Disadvantages:
 Penetration is slow; hence tissue sections should
not be more than 1 cm thick.
 It forms mercuric chloride deposits.
 It does not allow frozen tissue sections to be made.
 It inhibits the determination of the extent of tissue
decalcification.
ALDEHYDE FIXATIVES

Paraformaldehyde
 Polymerized form of formaldehyde (white powder
that causes turbidity).
 Suitable for paraffin embedding and sectioning,
and for immunocytochemical analysis.
 For long term tissue storage and good tissue
penetration.
ALDEHYDE FIXATIVES

Karnovsky’s Fixative
 Mixture of paraformaldehyde and
glutaraldehyde.
 Suitable for preparing samples for light
microscopy in resin embedding and sectioning,
and for electron microscopy.
 This fixative should always be fresh.
ALDEHYDE FIXATIVES

Glutaraldehyde
 It is made up of two formaldehyde residues,
linked by three carbon chains.
 It preserves cellular structures better; hence, it is
well – suited for electron microscopy work.
ALDEHYDE FIXATIVES

Glutaraldehyde
Advantages:
 Has a more stable effect on tissues.
 It preserves plasma proteins better and produces
less tissue shrinkage.
 More pleasant and less irritating to the nose and
does not cause dermatitis.
ALDEHYDE FIXATIVES

Glutaraldehyde
Disadvantages:
 It is more expensive and less stable.
 It penetrates tissues more slowly.
 It tends to make tissue (e.g. renal biopsy) more
brittle.
 It reduces PAS positivity of reactive mucin (like
10% NBF).
FIXATION (Part II)
Medical Laboratory Science Program
College of Allied Medical Professions
Lyceum of the Philippines University-Batangas
ALCOHOL FIXATIVES
Precipitating fixatives
 Not routinely used for tissues because they cause
too much brittleness and hardness unless studying
nucleic acids.
 Routinely used for cytologic smears because they
act quickly and give good nuclear detail.
 Mode of action: alcohols rapidly denatures and
precipitates proteins by destroying hydrogen and
other bonds thereby reducing the solubility of
proteins thereby stabilizing them.
 Generally can act both as a fixative and as a
dehydrating agent.
ALCOHOL FIXATIVES
100% Methyl alcohol (Methanol)
Advantages:
 It is excellent for fixing dry and wet smears, blood
smears and bone marrow tissues.
 It fixes and dehydrates at the same time.
Disadvantages:
 Penetration is slow.
 If left in fixative for more than 48 hours, the
tissues maybe over hardened and difficult to cut.
ALCOHOL FIXATIVES

95% Isopropyl alcohol

 Used for fixing touch preparations, although some
touch preparations are air dried and not fixed, for
certain special staining procedures such as Wright
– Giemsa.
ALCOHOL FIXATIVES

Ethyl alcohol
 Fixation time: 18 – 24 hours
 It is used at concentrations of 70 – 100%
 If used at lower concentrations, the RBCs become
hemolyzed and WBCs are inadequately
preserved.
 It may be used as a simple fixative but more
frequently incorporated into compound fixatives
for better results.
ALCOHOL FIXATIVES
Ethyl alcohol
Advantages:
 It preserves but does not fix glycogen.
 It preserves nucleoproteins and nucleic acids.
Disadvantage:
 Alcohols dissolve fats and lipids as a general rule.
Alcohol – containing fixatives are contraindicated
when lipids are to be studied.
ALCOHOL FIXATIVES
Carnoy’s Fixative (most rapid fixative)
 Absolute alcohol + chloroform + GAA
 Fixation time: 1 – 3 hours

Advantages:
 It permits good nuclear staining and
differentiation (because of GAA).
 It preserves Nissl granules and cytoplasmic
granules well.
 It is an excellent fixative for glycogen since
aqueous solutions are avoided.
Disadvantage: used for small pieces only.
ALCOHOL FIXATIVES

Clarke’s solution
 Ethanol + GAA
 Fixation time: 3 – 4 hours
 Has been used on frozen section and smears.
 Using conventional methods, fixation time should
be kept short.
 It preserves nucleic acids but extracts lipids.
ALCOHOL FIXATIVES

Alcoholic formalin
 HCHO + ethanol
 Fixation time: 12 – 24 hours
 Acts as fixative and dehydrating agent at the same
time.
 It clears and extracts lipids and therefore can be
used to visualize lymph nodes in breast tissues.
ALCOHOL FIXATIVES

Formol – acetic alcohol

 HCHO + ethanol + GAA
 Fixation time: 1 – 6 hours
 It is a faster acting agent than an alcoholic
formalin due to the presence of acetic acid.
ALCOHOL FIXATIVES
Gendre’s fixative
 HCHO + ethanol (saturated with picric acid) +
GAA
Advantages:
 Fixation is faster (fixation time is halved)
 Fixes and dehydrates at the same time.
Disadvantage:
 Produces gross hardening of tissues.
ALCOHOL FIXATIVES
Newcomer’s Fluid
 Isopropyl alcohol is the main component.
 Fixation time: 12 – 18 hours at 3oC.
 It is recommended for fixing
mucopolysaccharides and nuclear proteins.
 It produces better reaction in Feulgen stain (stain
for DNA) than Carnoy’s fluid.
 It acts both as a nuclear and histochemical
fixative.
METALLIC FIXATIVES

 Employs the use of the metal, mercury.

 Increases staining brightness and gives excellent
nuclear detail.
 Best fixative for hematopoietic and
reticuloendothelial tissues.
MERCURIC CHLORIDE FIXATIVES
 Most common metallic fixative frequently used in
saturated aqueous solutions of 5 – 7%
 They precipitate all proteins.
 It has greater affinity to acid dyes and is preferred in
lieu of formaldehyde for cytoplasmic staining.
 it is the routine fixative of choice for the preservation
of cell detail in tissue photography.
 Major disadvantage: It leads to the formation of
black granular deposits in the tissues. REMEDY 
Use saturated iodine solution in 95% alcohol
(alcoholic iodine).
MERCURIC CHLORIDE FIXATIVES

Zenker’s Solution
 Composition: (HIGAKANA ZENKI) 
*HgCl2
*Glacial acetic acid
*K2Cr2O7
*NaSO4
 Fixation time: 12 – 24 hours
MERCURIC CHLORIDE FIXATIVES

Zenker’s Solution
Advantages:
 It permits brilliant staining of nuclear and
connective tissue fibers
 Compatible with most stains
 Act as mordant to make certain special staining
reactions possible.
MERCURIC CHLORIDE FIXATIVES

Zenker’s Solution
Disadvantages:
 Penetration is poor.
 It causes lysis of RBCs and removes iron from
hemosiderin.
 It does not permit cutting of frozen sections.
 It has the tendency to form mercuric pigment
deposits or precipitate.
MERCURIC CHLORIDE FIXATIVES

Zenker’s Solution
 Precaution: Mercuric deposits may be removed by
immersing tissues in alcoholic iodine solution
prior to staining through a process known as de-
zenkerization
MERCURIC CHLORIDE FIXATIVES
Zenker – Formol (Helly’s Solution)
 Zenker’s Solution + HCHO
 Fixation time: 4 – 24 hours
 It is an excellent microanatomic fixative for
pituitary gland, bone marrow and blood
containing organs such as spleen and liver.
 Disadvantage: Same with Zenker’s solution but
only occurs when tissues are allowed to stay in the
fixative for more than 24 hours.
MERCURIC CHLORIDE FIXATIVES

Lillie’s Solution (B – 5 Fixative)

 Most commonly used for bone marrow biopsies.
 HgCl2 + Sodium acetate+ distilled water = stock
solution
 To use, stock solution + HCHO.
 Fixation time: 4 – 8 hours
MERCURIC CHLORIDE FIXATIVES
Heidenhain’s Susa Solution
 It is recommended mainly for tumor biopsies
especially on the skin; it is an excellent cytologic
fixative.
 Fixation time: 3 – 12 hours
 Composition: (HIGAKANAF SUSA) 
*HgCl2
*Glacial acetic acid
*K2Cr2O7
*NaCl
*Formalin
MERCURIC CHLORIDE FIXATIVES
Heidenhain’s Susa Solution
Advantage:
 Susa fixed tissues may and should be transferred
directly to 95% alcohol or absolute alcohol (to
avoid swelling).
Disadvantages:
 RBC preservation is poor.
 Mercuric chloride deposits tend to form on
tissues.
 Weigert’s method of staining elastic fibers is not
possible.
MERCURIC CHLORIDE FIXATIVES

Schauddin’s Sublimated Alcohol

HgCl2 + Methanol
It is used for smears of loose connective
tissues.
OXIDIZING AGENTS

Includes permanganate fixatives,

potassium dichromate, chromic acid and
osmium tetroxide.
They are used mainly as a secondary
fixative.
OXIDIZING AGENTS
Osmium Tetroxide (OsO4) or Osmic acid
 Pale yellow powder
 Traditionally used in electron microscopy both as a
fixative and a heavy metal stain.
 Penetration rate: 0.5mm/hour
 It fixes conjugated-fats and lipids permanently. Fats
formed hydrated osmium tetroxide are stained black
and easy to identify.
 Disadvantage: irritating to the eyes producing
conjunctivitis or causes deposition of black osmic
oxides in the cornea, producing blindness.
OXIDIZING AGENTS
Flemming’s Solution
 Most common chrome – osmium acetic acid
fixative used, recommended for nuclear
preparation of such sections.
 Chromic acid + Osmium tetroxide + GAA
 Fixation time: 24 – 48 hours
 It is an excellent fixative for nuclear structures
(e.g chromosomes).
 NOTE: Removal of GAA in the solution improves
the cytoplasmic detail of the cell.
 CHROMATE
FIXATIVES
Chromic acid
 It is most commonly used in 1-2% aqueous
solution usually as a constituent of a compound
fixative.
 It precipitates all proteins and adequately
preserves carbohydrates.
 NOTE: It is a strong oxidizing agent; hence, a
strong reducing agent (e.g. formaldehyde) must be
added to chrome – containing fixatives before use.
 CHROMATE
FIXATIVES

Potassium Dichromate
 It is most commonly used in a 3% aqueous
solution.
 It fixes but does not precipitate cytoplasmic
structures; preserves lipids and mitochondria.
 CHROMATE
FIXATIVES
Regaud’s (Muller’s) Fluid
 K2Cr2O7 + HCHO
 Fixation time: 12 – 48 hours
Advantages:
 It penetrates tissues well
 It is recommended for demonstration of
chromatin, mitochondria, mitotic figures, Golgi
bodies, RBC and colloid-containing tissues.
 CHROMATE
FIXATIVES
 Regaud’s (Muller’s) Fluid
Disadvantages:
 It deteriorates and darkens on standing due to
acidity.
 Prolonged fixation blackens tissue pigments.
 Glycogen penetration is poor. It is therefore
generally contraindicated for carbohydrates.
 CHROMATE
FIXATIVES
Orth’s Fluid
 K2Cr2O7 + HCHO + NASO4
 Fixation time: 36 – 72 hours
 It is recommended for study of early degenerative
processes and tissue necrosis
 It is used to demonstrate Rickettssiae and other
bacteria.
 PICRIC ACID
FIXATIVES
 An explosive hazard in dry form.
 It is a yellow colored solution which also dyes the
tissues. Hence, it acts as a fixative and a dye at the
same time. REMEDY  washing with changes of
50% and 70% ethanol.
 It preserves glycogen well.
 Picric acid fixed tissues must never be washed in
water before dehydration (picric acid creates
water soluble proteins) and should be transferred
or washed directly with 70% alcohol (p. 128, 4 –
5).
 PICRIC ACID
FIXATIVES

Bouin’s Solution
 Picric acid + HCHO + GAA
 Fixation time: 4 – 18 hours
 It is recommended for fixation of embryos and
pituitary biopsies.
 PICRIC ACID
FIXATIVES

Hollande’s Solution
 Picric acid + HCHO + GAA + Copper acetate +
distilled water
 Fixation time: 4 – 18 hours
 It is recommended for gastro – intestinal tract
specimens and fixation of endocrine tissues.
 PICRIC ACID
FIXATIVES

Gendre’s Solution
 HCHO + ethanol (saturated with picric acid) +
GAA
 It is highly recommended for the preservation of
glycogen and other carbohydrates.
 PICRIC ACID
FIXATIVES

Brasil’s Alcoholic Picroformol Fixative

 HCHO + picric acid + alcohol + TCA
 It is better and less “messy” than Bouin’s solution.
 It is an excellent fixative for glycogen.
 GLACIAL ACETIC
ACID
 Colorless liquid that freezes and solidifies at 16oC
hence the term “glacial”.
 Valuable for the preservation of nuclear
structures.
 Its major effect is to precipitate DNA, which is
split off from nucleoprotein.
 It is contraindicated for cytoplasmic fixation.
LEAD FIXATIVES

 They are used in 4% aqueous solution of basic

lead acetate.
 It is recommended for acid
mucopolysaccharides.
 TRICHLOROACETIC
ACID

It is used for the precipitation of proteins

and nucleic acids.
It is used as a fixative and a weak
decalcifying agent.
ACETONE

Not recommended as morphological

fixative for tissue blocks.
It is primarily used in fixing brain tissues
for the diagnosis of rabies.
 MICHEL’S
SOLUTION

It is a simple salt solution which provides

a stable transport medium of fresh
unfixed tissues such as renal, skin and
oral mucosa biopsies.
It is NOT a fixative.
FIXATION
General Precautions:
 All specimens should be properly labeled and
identified.
 Specimens should be fixed as soon as possible.
 If fixation is not immediately possible, refrigerate
samples but do not freeze.
 Fresh tissue may be infectious.
 Drying of specimens should be avoided.
 Distortion, rough handling and other mechanical
damage should be avoided.
FIXATION
General Precautions:
 Samples placed in NSS may cause autolysis.
 Tissues should not be more than 5 mm thick except
in lung edema ( 1 – 2 cm thick).
 Purulent materials, exudates and transudates
should be marked and kept for possible
examination.
 Fixatives should be 10 – 20 times the volume of the
tissue (Osmium tetroxides – 5 – 10 times).
 Museum fixation (50 times the tissue volume).
FIXATION
General Precautions:
 Contamination of fixed tissue with precipitates
should be avoided.
 Do not over fix tissues.
 Hollow organs should be packed with cotton
soaked with fixatives or opened before immersion.
 Air filled lungs may float. Cover the organ in
several layers of gauze to maintain the organ
under surface.
 Brain tissues maybe suspended with a cord to
prevent flattening.
FIXATION
General Precautions:
 Eyes should not be dissected before fixation. It
should be injected first with fixative.
 Frozen section may lead to formation of ice crystals
artifact.
 Muscle tissues maybe stretched by sutures or laid
flat in moist filter paper before suspending in
fixatives. This will prevent rigor contraction and
staining of artifacts.
 Water should NOT be used for glycogen containing
tissues (glycogen is water soluble).
FIXATION
General Precautions:
 Tissues maybe minced to assure rapid access of
the fixative.
 Hard tissues maybe softened with 4% aqueous
phenol solution overnight after fixation for easier
sectioning.
 Presence of mucus retards fixation (REMEDY -
wash with NSS).
 Fatty tissues should be cut in thin sections and
fixed longer.
FIXATION
General Precautions:
 Blood in tissues should be flushed with NSS before
fixation.
 The position or shape that is desired for sectioning
should be maintained before fixation.
 The fixative should penetrate from all sides.
 Hollow areas and natural cavities should be opened to
allow immediate access to fixatives.
 Gentle agitation (swirling) facilitates penetration.
 Use fresh and reagents of good quality.
FIXATION
General Precautions:
 If a specimen is received in fixative of dubious
quality, it must be replaced with a fresh fixative.
 Fixatives should be used only once.
 Avoid metal lids.
 Some fixatives require that specimens be washed
in water prior to processing.
 All fixatives are toxic and irritant.
FIXATION
General Precautions:
 Fixation is retarded by:
 Size and thickness of the tissue specimen –
larger tissues requires more fixatives and
longer fixation time.
 Presence of mucus – prevents complete
penetration of fixative. Wash with NSS.
 Presence of fats – should be cut in thin sections
and fixed longer.
 Presence of blood – wash with NSS
 Cold temperature – inactivates enzymes.
FIXATION
General Precautions:
 Fixation is enhanced by:
 Size and thickness of the tissue specimen
– smaller tissues requires less fixatives
and shorter fixation time.
 Gentle agitation
 Moderate heat
FIXATION
Difficulties caused by improper fixation:
 Failure to arrest early autolysis of cells
 CAUSES: failure to fix immediately, drying of
tissues, insufficient fixative
 Removal of substances soluble in fixing agent
 CAUSE: wrong choice of fixative
 Presence of artefactual pigments on tissue
sections.
 CAUSE: incomplete washing of fixative
FIXATION
Difficulties caused by improper fixation:
 Tissues are soft and feather – like in consistency.
 CAUSE: incomplete fixation
 Loss or inactivation of enzymes needed for study.
 CAUSE: wrong choice of fixative
 Shrinkage and swelling of cells and tissue structure.
 CAUSE: over-fixation
 Tissue blocks are brittle and hard
 CAUSE: prolonged fixation
FIXATION
Other considerations in fixation:
 Lipid fixation – commonly employs cryostat or frozen
sections for demonstrating lipids followed by a lipid
stain.
 Carbohydrate fixation – alcoholic fixatives are
generally recommended for glycogen fixation.
 Protein fixation – neutral buffered formal saline or
formaldehyde are commonly used.
 Electron microscopy – routine studies use
glutaraldehyde and osmium tetroxide (ultrathin
sections).
FIXATION
Other considerations in fixation:
 Enzyme histochemistry – 4% formaldehyde or
formal saline is used.
 Immunofluorescence – commonly uses formalin.
 Immunohistochemistry – employs organic
solvents (alcohols and acetone) and cross linking
reagents (paraformaldehyde).
 Microwave fixation – tissues are heated right
through the block in a very short time allowing
the study of cellular processes that proceed very
rapidly.
THANK YOU!