TYPES OF FIXATIVES/METHODS

OF FIXATION.

BY
SCT. SARATU UMAR (MRS)

COURSE CODE: HT 024

 OUTLINE
INTRODUCTION
 FACTORS AFFECTING THE QUALITY OF FIXATION
PROPERTIES OF A GOOD FIXATIVE
METHODS OF FIXATION
TYPES OF FIXATIVES
MOST IMPORTANT FIXATIVE IN HISTOPATHOLOGY
CONSTITUTION OF FORMALINE SOLUTIONS
FORMALIN PIGMENT
 OTHER MODES OF FIXATION
 INTRODUCTION
As soon as cells or tissues are removed from the body
they begin to die and undergo post-morterm changes.
These changes may be autolytic or putrefactive.
 Fixation prevents autolysis and putrefaction.
 If certain liquids and gases are added to the tissues, they
remain as lifelike as possible and autolysis and
putrefaction are prevented.
 INTRO CONT’D
 These liquids and gases are called fixatives.
 Examples of liquid fixatives are
 Formal saline,
 Acetone
 Methanol.
 Those in gaseous form are
 Formaldehyde,
 Acetaldehyde,
 Glutaraldehyde
 Acrolein.
 FACTORS THAT AFFECTS THE QUALITY OF FIXATION

 PH and buffer
 Duration of fixation and the size of specimens
 Temperature of fixative
 Concentration of fixative
 Osmolarity of fixatives and ionic composition
 Additives e.g calcium chloride, potassium dihydrogen phosphate
 PROPERTIES OF A GOOD FIXATIVE

 In addition to prevention of autolysis and putrefaction, a good

fixation should;
 Penetrate tissue rapidly
 Kill the tissue rapidly
 Prevent osmosis and leaching of cell and tissue constituents
 Allow accurate histochemistry of tissue constituents
 Give good optical differentiation to unstained tissue
constituent for easy examination under the microscope
 Harden tissue for easy handling
 Support high quality and consistent staining with H&E.
 METHODS
OF FIXATION

PHYSICAL

Heating
Microwaving
Freeze drying
 METHODS OF FIXATION
Heat fixation
Perfusion
Immersion
Vapour method
Spray/coating
PRINCIPLE OF ACTION OF
FIXATIVES (ALDEHYDE GROUP).
Fixatives acts by denaturing or precipitating proteins
which then form a meshwork due to cross linking of
proteins.
This meshwork tends to hold the other cell constituents
in vivo relation to each other and insoluble proteins
provide mechanical strength for subsequent procedures.
 TYPES OF FIXATIVES

Compound Simple

Micro- Cytologi Histoche

anatomical cal mical
 SIMPLE FIXATIVE
A solution or a gas, which contains only one fixative is called
simple fixative.
Examples are
 10% Formalin
 10% Formal saline,
 Buffered formalin
 Glutaraldehyde
 Mercuric Chloride (HgC12)
 Potassium Dichromate (K2Cr207)
 Picric Acid (C6H2(NO2)3OH)
 Osmium Tetraoxide (OSO4)
 FORMALDEHYDE (HCHO)

 Formalin is the most important and most used fixative in

histopathology and histochemistry laboratories.
 Formaldehyde is a gas and it is a product of oxidation of
methanol.
 It is soluble in water to about 40%.
 When the gas is dissolved in water, it is called formalin.
 The dry powder or pellet form of formaldehyde is called
paraformaldehyde and can be dissolved in water by heating to
600c
 FORMALDEHYDE (HCHO)…….

 For routine work, formalin is used at a concentration of 10%

formalin (4% formaldehyde), which may be in a buffered solution
or in an isotonic saline solution.
 Stored formalin soon oxidizes to yield small amount of formic
acid, which reduces the pH of the solution.
 To prevent this, commercial formalin contains methanol which
acts as a preservative, although, this prevents its use for electron
microscopy work.
PRINCIPLE OF FORMALIN FIXATION
 Formalin acts by polymerizing action, the formation of
complexes by development of links (methylene bridges)
between protein molecules.
 ADVANTAGES
 It fixes evenly and produces little shrinkage of tissue
 It supports many staining techniques
 Optimizes antigen retrieval step in
immunohistochemistry.
 Museum specimens are better fixed in it.
 Molecular techniques such as ISH have also been
validated for use on formalin fixed tissue
 DISADVANTAGES
 It produces formalin pigment in blood containing organs
although, this pigment is not found in tissue fixed in buffered
formalin
 Formalin is a source of hazard to health.
 Flammability
 The practical and legal requirements of disposal after use.
 The vapour causes eye irritation and sinusitis.
 When prolong contact with the skin, it causes dermatitis.
 COMMON FORMALIN SOLUTIONS
 10% FORMALIN
CONSTITUENT:
Tap water-------------------------------90mls
Formaldehyde (37-40%)-------------------10mls
 10% FORMOL SALINE
CONSTITUENT:
Tap water--------------------------------90mls
Formaldehyde (37- 40%)--------------------10mls
Sodium chloride----------------------------0.9g
10% NEUTRAL BUFFERED FORMALIN (100mls) (ph
7.0)

CONSTITUENT:
Tap water-------------------------------90mls
 Formalin(37-40% formaldehyde)----------10mls
 Sodium chloride------------------------0.85g
 Disodium hydrogen phosphate------------0.65g
 Sodium hydrogen phosphate/disodium phosphate...
……………………………..0.45g
 MINIMUM TIME REQUIRED FOR
FIXATION
 8 Hours at room temperature

 4 Hours may be sufficient with agitation.

 Fixation time can be reduced to 25-40% by increasing

the temperature to 45oC
 FORMALIN PIGMENT
 Is the artefactual pigments deposited when formalin
solution is used as a fixative due to the formation of a
compound known as acid formaldehyde hematein.
 The compound is formed when formalin which is acidic
in pH comes in contact with a blood tissue.
 This material found in tissues that have been fixed in
simple formalin fixatives, as these solutions age, formic
acid developes from the formaldehyde content and lowers
the pH.
FORMALIN PIGMENT CONT’D
 This in turn causes crystals of formalin pigments or
acid formaldehyde hematein to be deposited
throughout the tissues.
 Formalin pigment appears as a
 brown,
 granular,
 double refractile deposit seen both intracellularly and
extracellularly on tissue microscopically
REMOVAL OF FORMALIN PIGMENT
 It may be removed from sections prior to staining quite
easily by
  Saturated picric acid

 Kardasewitsch’s method

 Lillies method

 1% sodium hydroxide in absolute ethanol

 COMPOUND FIXATIVES
 When two or more simple fixatives are combined in a
solution, resulting solution is called a compound fixative.
Such a solution either
 combines the advantages of the constituent fixative as in
Zenker’s formol which contains mercuric chloride, potassium
dichromate and formalin or
 act against each other so as to neutralize the disadvantages of
the constituent fixative
 E.g acetic acid which swells tissue is combined with picric
acid which shrinks tissue in Bouin’s fluid.
 MICRO-ANATOMICAL FIXATIVE

Fixative which preserve the architecture of tissues and enable

the relationship between cells and tissue substances to be
maintained, are called micro-anatomical fixatives.
Majority of them are compound fixatives but the most used of
them, 10% formol saline which is also a simple fixative.
EXAMPLES OF MICROANATOMICAL

1O% Formal saline

Buffered formalin
Heidenhain’s Susa
Bouin’s Fluid
Formol Sublimate
Zenker’s Solution
Helly’s Fluid
 CYTOLOGICAL FIXATIVE

 Cytological fixatives, are chemicals which preserve cell

constituents,
 Maintain nuclear and cytoplasmic substance for histochemistry
and other cytological procedures
 There are two types of cytological fixatives.
 Nuclear fixatives
 Cytoplasmic fixatives.
Some alcohols are specifically recommended for fixation
of cytological preparations,95% ethanol/ethyl
alcohol,100% methanol, isopropyl alcohol/propanol ,1:1
alcohol ether.
EXAMPLES OF CYTOLOGICAL
FIXATIVES.

CYTOLOGICAL FIXATIVE

NUCLEAR FIXATIVE

Flemming’s fluid

Carnoy’s fluid

CYTOPLASMIC FIXATIVE

Flemming’s without acetic acid

Helly’s fluid
 HISTOCHEMICAL FIXATIVES
These are used to demonstrate the chemical constituents
of cell. E.g
Formal saline
Cold acetone
Absolute alcohol
OTHER MODES OF
FIXATION
 SECONDARY FIXATION
 Tissues removed from 10% formol saline may be transferred
to another fixative either because
 a more vigorous precipitant is desired or
 an improved preservative property of certain tissues
constituents is desired.
 Fixative commonly used in secondary fixation are
 Helly’s fluid
 Zenker’s fluid.
 POST FIXATION

When cells and proteinous substances are enveloped in a fatty

part of the tissue, they are not adequately fixed because
fixatives, which are aqueous solutions, do not penetrate fats
effectively.
During tissue processing, the clearing agent removes the fat.
 Hence an unfixed region of cells and protein rich substances is
exposed.
 To prevent degradation of tissue, which may occur, tissue
should be transferred from the clearing agent back to absolute
alcohol for some hours where it is post fixed.
Tissue is then returned to the clearing agent, paraffin wax and
embedded in paraffin wax.
 POST CHROMATIZATION

 This is also called post chroming or post mordanting.

 It is the treatment of tissue with 3% potassium dichromate after

the primary fixative which is usually 10% formol saline.
 Post chromatization is done to mordant tissue when demonstrating
mitochondria in the Alteman’s method and for myelin in the
Weigert-Pal’s techniques.
 Post chromomatization can be performed in two ways.
 On tissues before processing and
 On section before staining
 FIXATION OF WHOLE ORGANS

 The reagent of choice for the fixation of whole organs from where
tissues are to be processed and sectioned is 10% formol saline.
 However, this may be followed by secondary fixation if a more
vigorous fixative is required.
 EXAMPLES OF ORGANS
 Heart,
 intestine,
 uterus,
 Brain
 eye,
 liver
 Pancreas and kidney
 EMBALMING

Embalming is the art and science of preserving human or animal

remains by treating them (in its modern form with chemicals) to
forestall decomposition .
The intention is usually to make the deceased suitable for public
or private viewing as part of the funeral ceremony, or keep them
preserved for medical purposes in an anatomical laboratory.
Embalming chemicals are a variety of preservatives, sanitizers,
disinfectant agents, and additives used in modern embalming to
temporarily delay decomposition and restore a natural appearance
for viewing a body after death.
 EMBALMING CONT’D…….
 Typical embalming fluid contains a mixture of

 Fixative- eg alcohol and formaldehyde,

 Phenol
 Glycerine.
 The formaldehyde content generally ranges from 5 to 35
percent
 The ethanol content may range from 9 to 56 percent.
 SUMMARY

Fixation of tissue is a process by which the cells and tissue are fixed
in chemicals and partly physical state so that they can withstand
subsequent treatment with various reagents.
 Fixation results in denaturation and coagulation of protein in the
tissues.
Penetration rate differs in different fixative.
An ideal fixative should me cheap,penetrate tissue rapidly,kill tissue
rapidly.
 Immersion, perfusion,vapour,coating/spray,freeze
drying,microwave fixation are the different methods of fixation.
 SUMMARY CONT’D
The most commonly used method is the simple immersion of
tissues in an excess fixative.
Bufferd formalin is the most commonly used fixative and it
prevents pigment formation on tissue.
3% potassium dichromate is used as secondary fixative during
post chromatization.
CONCLUSION
Fixation is considered as key step in histopathology pro-
cedure. Each and every fixative has its own advantage
and
disadvantage. Various different fixatives perform various
functions, and various factors such as size, temperature,
and
osmolarity have direct effect on fixation procedure.
Using the appropriate fixative is necessary to
ensure the most significant histologic features are
highlighted while not interfering with or
precluding ancillary testing that may be required.
THANK YOU FOR
LISTENING.
 QUESTIONS

1. Which is not the aim of fixation?

A. To prevent putrefaction
B. Remove excess water from the tissue
C. Prevent osmotic swelling
D.Render the tissue suitable for subsequent staining
E. To prevent lysosomal activity
 2. Which statement about Formaldehyde is false?
A. Most commonly used Aldehyde fixative
B. It is a water soluble gas (sat. approx. 40%)
C. Concentrated formaldehyde is 40%
D. Penetrates tissue rapidly
E. Fixes tissue rapidly
F.The best fixative for nucleic acids
 3. Which statement about Glutaraldehyde is false?
A. Widely used in EM
B. Gives a better morphological picture at the EM level
C. Cross-links less extensively than formaldehyde
D. Diffuses into tissue very slowly
E. Fixes tissue components rapidly
F. Renders tissue too hard for paraffin sectioning
4 Formalin Solution (10%, unbuffered) is composed of:
A. 10ml Formaldehyde (40%) and 90ml distilled water
B. 50ml Formaldehyde (40%) and 50ml distilled water
C.90ml Formaldehyde (10%) and 10ml distilled water
D. 90ml Formaldehyde (10%) and 10ml distilled water
 5. Which is not an Aldehyde fixative?
A. Picric Acid
B. Potassium Dichromate
C. Glutaldehyde
D. Mercuric chloride
6. Why isn't microwave fixation suitable for large specimens (of tissues/organs)?
A. Microwave fixation is best suited on tissue blocks 10-12mm thick
B. The microwave radiation can damage fine tissue structures
C. It is unable to heat the centre of the tissue/organ sufficiently w/o overheating the
outside
D. Using microwaves accelerates the rate of putrefaction
 7. Acetic acid...
A. Is a denaturing agent
B. Swells collagen
C. Is a percipitating fixative
D. Is an alcohol
E. Is all of the above
8. Which is NOT an essential of good fixation?
A. Fix it ASAP
B. Refrigerate it if fixation isn't immediately possible
C. Reagents are carefully made up
D. Fixative is recycled and reused
E. A suitable treatment follows fixation
 9 Mercuric Chloride is restricted in use because:
A. It's toxic
B. Its corrosive
C. Its a pollutant
D. All of the above
10. .................... prevents the formation of formalin
pigment