First and most critical step in histotechnology
Classically defined as killing, penetration and
hardening of tissue.
Currently define as alteration of tissues by
stabilizing proteins so that tissues become
resistant to further changes.
Primary aim: to preserve the morphologic and
chemical integrity of the cell in as life-like manner
as possible.
Secondary aim: to harden and protect the tissue
from trauma of further handling
Preserve the tissue
Stop all cellular activities so that cells can be
viewed microscopically as if they are still in living
state.
Prevent breakdown of cellular elements
Autolysis - self digestion
Putrefaction - further decomposition after death
Coagulate or precipitate Protoplasmic
Substances
Denaturation
Change soluble substance to insoluble structures that
may leak during further handling
Hardens tissue allowing thin sectioning
Prevent autolysis and inactivated infectious
agents
Improves cell avidity for special stains
Additive Fixation
Alter tissue by bonding with it and adding themselves
to the tissue by forming cross link/complexes giving
stability
e.g. Formalin, Mercury
Non-Additive Fixation
Act on tissue without combining with it, but alters
and stabilize tissue by removing bound water
causing shrinkage and hardening.
e.g. Alcohol
Hydrogen Ion Concentration (pH)
pH 6-8
Ultrastructural preservation
Temperature
temp fixation time
Room temperature for satisfactory result (40C).
Specimen size/Thickness
1-2 mm2 (electron); 2 cm2 (light microscopy); 2-3
mm thickness
Large Tx should be opened/sliced thinly
Volume ratio
Recommended = 1:10
Ideal = 1:15-20
Osmolality
Recommended = slightly hypertonic solution (400-450
mOSm).
Concentration
Lowest level possible
Formaldehyde 10%; Glutaraldehyde 3%
Time
Cold ischemia time = within 20-30 minutes after
interruption of blood supply. (<60 min.)
Fixation time = 6-48 hours
According to Composition
Simple Fixatives
One component substance
Aldehydes, Oxidizing agents, Metallic, Alcohols
Compound Fixatives
2 or more fixatives to obtain optimal combined effects.
According to Action
Microanatomical general microscopic study of
tissue structures
Cytological Fixatives specific parts and particular
microscopic elements of the cell
Nuclear Fixatives with Glacial Acetic Acid
Cytoplasmic Fixatives without Glacial Ascetic Acid
Histochemical Fixatives preserves chemical
constituents
Crosslinking Fixatives
Creates covalent bonds between proteins and
tissue providing additional rigidity
E.g. Aldehydes
Precipitating Fixatives
Reduce protein solubility by disrupting
hydrophobic interaction
E.g. Alcohols
FORMALDEHYDE
37-40% - stock solution (formaldehyde)
10% - working solution (formalin)
Widely used fixative
Fixation time: 24 hours
ADVANTAGES DISADVANTAGES
Cheap, readily available Irritating fumes
Compatible with many Formation of pigments (if
stains unbuffered)
Preserves FAT, MUCIN,
GLYCOGEN and PROTEINS
10% Formal Saline
Microanatomical fixative
CNS Tissues, Tissues for Histochemical preparations
Composition:
Formaldehyde 100 mL
Sodium dihydrogen phosphate monohydate 4g
Disodium hydrogen phosphate anhydrous 6.5 g
D. H2O 900 mL
10% Neutral Buffered Formalin (10% NBF)
Best general fixative
Prevents formation of acid formalin pigments
Composition: (pH 7.4)
NaH2PO4 6.5 g
NaH2PO4.H2O 4.0 g
Formaldehyde 100 mL
D. H2O 900 mL
Zinc Formalin
Alternative to mercuric chloride preparations
Composition:
Zinc sulphate 1g
Deionized water 900 mL
Formaldehyde 100 mL
Formol-Corrosive (Formal Sublimate)
Routine post-mortem tissues
Composition:
Mercuric chloride 90 mL
Formaldehyde 10 mL
Inhibits determination of the extent of tissue decalcification
PARAFORMALDEHYDE
Polymerized form of Formaldehyde
Routine paraffin embedding and sectioning and
immunocytochemical analysis.
Karnovskys Fixative
Samples for resin embedding and sectioning
Electron microscopy
Composition:
8% Paraformaldehyde 25 mL
25% glutaraldehyde 10 mL
0.2 M PO4 buffer 50 ml
D. H2O 100 mL
GLUTARALDEHYDE
Electron microscopy
Glutaraldehyde
10% NBF
Osmium tetroxide
Platinic chloride (PtCl3)
Platinic chloride-Formalin (Zambonis
Fixative)
Gold Chloride (AuCl)
Used as fixative and dehydrating agent
Glycogen preservation
Contraindicated for lipids and fats
Methyl Alcohol (100%)
Fix dry and wet smears, blood smears and bone
marrow tissues
Isopropyl Alcohol (95%)
Fix Touch Preparations
Ethyl Alcohol
Frequently incorporated into compound fixatives
Carnoys Fluid
Fix chromosomes, lymph glands and urgent biopsies
Brain tissues for diagnosis of rabies
most rapid fixative
Composition:
Absolute alcohol 60 mL
Chloroform 30 mL
Glacial Acetic acid 10 mL
Clarkes Solution
Frozen sections and smears
Composition:
Ethanol 75 mL
Glacial acetic Acid 25 mL
Alcoholic Formalin
Fixation or post-fixation of large fatty specimen
(breast)
Composition:
Formaldehyde 100 mL
Ethanol (95%) 900 mL
Formol-acetic acid
Fix diagnostic cryostat sections
Composition:
Ethanol (Abs) 85 mL
Formaldehyde 10 mL
Glacial Acetic acid 5 mL
Gendres Fixative (Alcoholic Formalin)
Alcoholic Boiuns solution
Preserves glycogen and other carbohydrates;
Immunoperoxidase studies
Composition:
95% Ethyl Alcohol saturated with Picric acid 80 mL
Formaldehyde 15 mL
Glacial Acetic Acid 5 mL
Rapid diagnosis (Fixative + Dehydrant)
Newcomers Fluid
Nuclear and Histochemical fixative
Composition:
Isopropyl alcohol 60 mL
Propionic acid 30 mL
Petroleum Ether 10 mL
Acetone 10 mL
Dioxane 10 mL
MERCURIC CHLORIDE
Most common metallic fixative
Used as secondary fixative
Recommended for RENAL TISSUES, FIBRIN,
CONNECTIVE TISSUE and MUSCLES
Zenkers Fluid
small tissues of liver, spleen, connective tissue.
Composition:
Mercuric chloride 5g
Potassium dichromate 2.5 g
D. H2O 100 mL
Glacial Acetic acid 5 mL (before use)
Hellys Solution (Zenkers Fomol )
Pituitary gland, BM, blood containing organs
(liver, spleen).
Composition:
Mercuric chloride 5g
Potassium dichromate 2.5 g
D. H2O 100 mL
Formaldehyde 5 mL
Lillies B-5 Fixative
Bone marrow biopsies
Composition: (Stock)
Mercuric chloride 12 g
Sodium acetate 2.5 g
D. H2O 200 mL
working solution
B-5 Stock 20 mL
Formaldehyde 2 mL
Heidenhains Susa Solution
Tumor biopsies, Cytologic Fixative
Composition:
Mercuric chloride 45 g
NaCl 5g
Trichloroacetic acid 20 g
Glacial Acetic acid 40 mL
Formaldehyde 200 mL
D. H2O 800 mL
Reacts with various side chains of proteins,
forming crosslinks that stabilize tissue
structure
Use as secondary fixatives
E.g. Permanganate, Chromates, Osmium
tetroxide
Fixes conjugated fats and lipids
Inhibits hematoxylin and makes
counterstaining difficult
Precaution: may cause conjunctivitis or
blindness.
Flemmings Solution
Recommended for Nuclear preparation
Composition:
Aqueous Chromic acid 15 mL
Aqueous Osmium tetroxide 4 mL
Glacial Acetic acid 1 mL
Flemmings Solution without Acetic acid
For cytoplasmic preparations
Composition:
Aqueous Chromic acid 15 mL
Aqueous Osmium tetroxide 5 mL
CHROMATE (CHROMIUM)
Chromic Acid
Constituent of compound fixative
Oxidizer (added to reducer)
Preserve CARBOHYDRATES, precipitate PROTEINS
Potassium dichromate
3% aqueous solution
Preserves LIPIDS, Mitochondria
Mullers (Regards) Fluid
Demonstration of chromatin, mitochondria,
mitotic figures, Golgi bodies, RBC and colloid-
containg tissues.
Composition:
Potassium dichromate 80 mL
Formaldehyde 20 mL
Orths Fluid
Demonstrate Rickettsiae and other bacteria
Composition:
Potassium dichromate 100 mL
Sodium sulfate 1g
Formaldehyde 10 mL
Highly explosive when dry
Produce excessive yellow staining of tissues
To remove yellow discoloration
Tissue 70% ethanol 5% Na thiosulfate water
Bouins Solution
Embryos and pituitary biopsies
Fragmentary biopsies
Composition:
Saturated Picric acid 75 mL
Formaldehyde 25 mL
Glacial Acetic acid 5 mL
Hollandes Solution
Gastro-intestinal tract specimen and endocrine
tissues
Declacifying properties
Composition:
Copper acetate 25 g
Picric acid 40 g
Formaldehyde 100 mL
Acetic acid 15 mL
D. H2O 1000 mL
Brasils Alcoholic Picroformol Fixative
Fixation of glycogen
Composition:
Formaldehyde 2040 mL
Picric acid 80 g
Ethanol/Isopropyl Alcohol 6000 mL
Trochloroacetic acid 65 g
Causes tissue to swell
Useful for nuclear study
Contraindicated for cytoplasmic fixation
(destroys mitochondria and golgi elements)
4% aqueous solution of Lead Acetate
Recommended for acid mucopolysaccharides
Trichloroacetic Acid
Swelling effect
Weak decalcifying agent
Acetone
Use for diagnosis of rabies
Study of water diffusable enzymes (phosphatase,
lipase)
Heat Fixation
Frozen tissue sections; bacterial smears
Direct flaming
Stable medium for transport of fresh, unfixed
tissues; which will undergo subsequent
FROZEN SECTION and
IMMUNOFLUORESCENCE studies.
Not a fixative, not recommended for flow
cytometry and fluorescent in-situ
hybridization assays.
Is the process of placing already fixed tissue
in a second fixative in order to:
Facilitate and improve the demonstration of
particular substances.
Make special staining techniques possible
To ensure further and complete hardening and
preservation of tissues.
A form of secondary fixation whereby a
primarily fixed tissue is placed in aqueous
solution of 2.5- 3% potassium dichromate for
24 hours to act as mordant for better staining
effects and to aid in cytologic preservation of
tissues.
The process of removing excess fixative from the
tissue after fixation in order to improve staining
and remove artifacts from the tissues.
Several solutions may be used:
Tap water used to remove excess Chromates,
formalin, osmic acid fixatives
50 70% alcohol - used to wash out picric acid fixatives
Alcoholic Iodine used to remove excess mercuric
fixatives
Formalin pigment
1. De-wax the sections, rinse in 100% alcohol,
rinse in 70% alcohol, rinse in distilled water.
2. Treat in saturated alcoholic picric acid for 30
minutes to 2 hours.
3. Wash well in running tap water.
4. If yellow staining of the section persists rinse
in dilute lithium carbonate.
5. Rinse in tap water.
Mercury pigment
1. De-wax the sections, rinse in 100% alcohol, rinse
in 70% alcohol, rinse in distilled water.
2. Treat in Lugol's iodine for 2 minutes.
3. Decolorize in 5% Sodium thiosulfate for 5
minutes.
4. Wash well in running tap water
Fixatives containing PICRIC ACID
1. Tissues fixed in non-alcoholic picric acid-based
fixatives are washed in repeated 1-3 hourly changes
of 50%-70% ethanol until the supernatant is faintly
yellowish or clear. This may take 2-3 days.
2. Specimens fixed in alcoholic picric acid fluids are
washed in 80%-90% ethanol, as anhydrous
conditions must be maintained.
3. Picric acid retained in tissues can impede wax
infiltration and exacerbate static electrification of
ribbons during sectioning. It also has an adverse
affect on stored wax embedded tissues.
Dichromate pigment
1. De-wax the sections, rinse in 100% alcohol, rinse
in 70% alcohol, rinse in distilled water.
2. Treat in 2% HCl in 70% alcohol or Dioxane16-24
hours.
3. Rinse in tap water.
AUTOLYSIS INCOMPLETE FIXATION
Minimize cold ischemia Increase fixation time
time Change fixative
Adequate ratio of Observe proper tissue
fixative to tissue thickness for better
Sectioning of large penetration
tissues prior to fixation Frequent change in
fixatives
Agitate
Structures to be stained Recommended Fixative Not Recommended
Fixative
Amyloid
Bacteria
Fats and Lipids
Negri Bodies
Melanin Pigment
Nervous Tissue
Polysaccharide
Nissl bodies
Bone Marrow
Samples for Electron
Microscopy
