Date post: | 29-Nov-2014 |
Category: |
Documents |
Upload: | khushboo-dewan |
View: | 149 times |
Download: | 8 times |
FIXATION AND FIXATIVES
AIMS OF FIXATION Prevention of putrefaction and autolysis
of tissues Prevention of change of shape or
volume during any subsequent procedures
Maintainance of tissues in a state close to their normal living state
Solidification of colloid material of cells and hardening of tissues
DISADVANTAGES OF FIXATION Molecular loss Swelling/shrinkage Variation in staining Accuracy of biochemical analysis
Capacity to maintain intracellular organelles
FACTORS EFFECTING FIXATION
pH Temperature Thickness of the tissue section
Osmolality of solution Duration of fixation additives
TYPES OF FIXATION PHYSICAL-HEAT - MICROWAVE - FREEZE DRYING
CHEMICAL
CLASSIFICATION
Simple Compound
Microanatomical fixativeCytological fixativeHistochemical fixative
REAGENTS EMPLOYED AS FIXATIVES Formaldehyde
40% formaldehyde= formalin cross links between proteins-?? fixes phosholipids and unsaturated fatty acids
Advantages- easily available cheap penetrates and fixes tissues well preserves fat, myelin, nerve fibres, amyloid, haemosiderin, organisms
FORMALIN Disadvantages paraformaldehyde slow dermatitis, irritating to eyes impurity of formic acid iron and other pigments changes colour of bile precipitates fine crystalline pigment
from hemoglobin REVERSIBILITY
GLUTARALDEHYDE MOA- cross link-?? Advantages- - ultrastructure Thickness of section IHC staining
MERCURIC CHLORIDE MOA- ammonium salts, amides, amines Rapid penetration and hardening Cytoplasmic and nuclear staining Poor depth of penetration Shrinkage Overhardening Radio-opaque Corrodes metals
POTASSIUM DICHROMATE Good cytoplasmic stain Preserves phospholipids Moderate hardening Mordant for staining Poor nuclear stain Wash in water overnight before transfer
to alcohol
CHROMIC ACID Powerful oxidising agent Preserves carbohydrates Orth’s fluid and zenker’s fluid
OSMIUM TETRAOXIDE 2% stock solution Lipids like myelin –black-?? Cytological fixation Expensive Poor and uneven penetration Irritating to eyes ultrastructure
PICRIC ACID Explosive when dry Precipitates proteins as picrates Trichrome stain gives a good contrast
ETHYL ALCOHOL Fixing blood smears Carnoy’s fluid Precipitates protein and glycogen is
preserved Demonstration of enzymes Dissolves fat and lipids
ACETONE Histochemical demonstration of
enzymes phosphatases and lipases
ACETIC ACID Swelling of tissues Good nuclear fixation Poor cytoplasmic fixation
TRICHLOROACETIC ACID Swells tissues Protein precipitant Decalcifying properties
MICROANATOMICAL FIXATIVESFORMAL SALINE 100 ml formalin 900 ml tap water 8.1 g NaCl Tolerant fixative 3 mm thick-56°C-3 hrs Neutral buffered formalin?? Formal zinc??
FORMAL CALCIUM 90 ml water 10 ml formalin 2 g calcium chloride preserves phosholipids Not recommended if calcium, acid or
alkaline phosphatases have to be estimated.
HEIDENHAIN SUSA HgCl2 NaCl Formalin Trichloroacetic acid Glacial acetic acid Distilled water Best fixative for routine biopsy Intolerant fixative Good cytological staining
ZENKER’S FLUID HgCl2 Potassium dichromate Sodium sulfate Distilled water Glacial acetic acid Bloody specimen Trichrome stain Mallory’s connective tissue stain
HELLY’S FLUID Zenker’s+formalin For hematologic tissues If giemsa/ leishman stains
BOUIN’S & GENDRE’S FLUID Picric acid Formalin Glacial acetic acid Connective tissue stain Glycogen preservation-RCC, liver, muscle
CYTOLOGICAL STAINSCARNOY’S FLUID
ABSOLUTE ALCOHOL CHLOROFORM GLACIAL ACETIC ACID RAPID NUCLEAR FIXATION GLYCOGEN PRESERVATION
NEWCOMER’S FLUID ISOPROPANOL PROPIONIC ACID PETROLEUM ETHER ACETONE DOXANE RAPID FIXING CHROMOSOME
CHAMPY’S FLUID POTASSIUM DICHROMATE CHROMIC ACIID OSMIUM TETRAOXIDE Slow uneven penetration Cytological fixation Preserves fat, lipids