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7/25/2019 Fixative s
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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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