Fixation Flashcards
first and most critical step in histotechnology
fixation
Primary aim of fixation
to preserve the morphologic and chemical integrity of the cell in as life
like manner as possible.
Secondary goal of fixation
- to harden and protect the tissue from the trauma of further handling,
– so it is easier to cut during gross examination
Fixation prevents __________ of tissues after removal from the body
degeneration, decomposition, putrefaction and
distortion
Fixatives stops all cellular activities
To preserve the tissue
Fixation prevents autolysis by inactivating the lysosomal
enzymes or by chemically altering, stabilizing and making the
tissue components insoluble
To prevent breakdown of cellular elements
2 Basic Mechanisms
- Additive fixation
- Non-additive fixation
example of additive fixation
Formalin, mercury, osmium
tetroxide
example of Non-additive fixation
Alcoholic fixative
Effects of Fixatives
*Preserve the morphologic and chemical integrity of the cell
*Harden soft & friable tissues
* Inhibit bacterial decomposition
*Act as mordants or accentuators
Main Factors Involved in Fixation
Osmolality
Concentration
Duration of fixation
Hydrogen ion concentration
Temperature
Thickness of section
ideal conc in Immuno EM
0.25%
temperature for electron microscope & Histochemistry
0-4⁰C
Formalin Heated to 60⁰C
rapid fixatio
Formalin at 100⁰C
fix tissues with tuberculosis
thickness of section
– 1-2 mm² for electron microscopy
– 2 cm² for light microscopy
example of Large solid tissue
Uterus
BRain
Practical Considerations of Fixation
Speed
Penetration
Volume
Duration of Fixation
Characteristics of fixation
*Cheap, stable, safe to handle
*Must be isotonic
* Inhibit bacterial decomposition
*Must permit rapid and even penetration of tissues
*Must make cellular components insoluble to hypotonic
solutions.
Types of Fixatives according to composition and according to action
according to composition:
- simple fixative
- compound fixative
according to action
- microanatomical fixative
- cytological fixative
- histochemical fixative
Simple Fixatives
- aldehyde
- metallic fixative
fixative under aldehyde
- formaldehyde
- glutaraldehyde
fixative under metallic
ØMercuric chloride
ØChromate fixatives
ØLead fixatives
ØHeat
fixative under Chromate fixatives
ØPotassium dichromate
ØChromic acid
fixative under Lead fixatives
ØAcetone
ØAlcohol
ØPicric acid
ØAcetic acid
ØOsmium tetroxide (Osmic acid)
Permit general microscopic study of tissue structures
Microanatomical Fixatives
Preserve specific parts
Nuclear
Cytoplasmic
Cytological Fixatives
Preserve the
chemical constituents of cells and tissues
Histochemical Fixatives
Microanatomical Fixatives
- 10% Formol Saline
- 10% Neutral Buffered Formalin
3.Heidenhein’s susa
4.Zenker’s solution
5.Zenker’s formol (Helly’s solution)
6.Bouin’s solution
7.Brasil soln
preserve nuclear structure
(chromosomes). Contain
glacial aa, pH 4.6 or less
Nuclear fixatives
preserve cytoplasmic structure, no glacial aa, pH > 4.6
Cytoplasmic Fixatives
Nuclear fixative
- Flemming’s Fluid
- Carnoy’s Fluid
- Bouin’s Fluid
- Newcomer’s Fluid
- Heidenhain susa
Cytoplasmic fixative
1.Flemming’s Fluid
without acetic acid
2.Helly’s Fluid
3.Regaud’s Fluid (Muller’s fluid)
4.Orth’s Fluid
Histochemical Fixatives
1.Formol Saline 10%
2.Absolute Ethyl Alcohol
3.Acetone
4.Newcomer’s Fluid
most widely used aldehyde fixative
10% Formaldehyde (formalin)
unsatisfactory for
routine fixation
Pure stock solution of 40%
A gas produced by the oxidation of methyl alcohol
formaldehyde
Preserves fats, glycogen and mucin
formaldehyde
Recommended for nervous tissue preservation
formaldehyde
Recommended for fixation of central
nervous tissues and general
postmortem tissues for histochemical
examination
1-% formol saline
Physical methods include
heating, microwaving and cryo-preservation (freeze drying)
May cause sinusitis, allergic rhinitis, excessive
lacrimation or allergic dermatitis
formaldehyde
Preserves enzymes and nucleoproteins
10% formol saline
Recommended for
preservation and storage
of surgical, post-mortem
and research specimen
10% NBF or phosphate bufferd formalin
recommended for routine postmortem
tissues
Formol – Corrosive or Formol – Sublimate
Penetration is slow
- Formol – Corrosive or Formol – Sublimate
- Glutaraldehyde
- Methyl Alcohol
Demonstrates fats and mucins
10% formol saline
Penetration is poor
- Zenker’s Fluid
- trichloroacetic acid
Best fixative for tissues
containing iron pigments
and for elastic fibers
10% NBF or phosphate bufferd formalin
Excellent for many staining
procedures including silver
reticulum methods
Formol – Corrosive or Formol – Sublimate
Considered as the most rapid
fixative
carnoy’s fluid
Used to fix sputum since it
coagulates mucus
Alcoholic Formalin or Gendre’s Fixative
For routine light microscopic work
Glutaraldehyde
Very expensive
- glutaraldehyde
- osmium tetroxide
Reduces PAS positivity of reactive mucin
-10% Neutral buffered formalin or
Phosphate-buffered formalin
- Regaud’s Fluid or Muller’s Fluid
Recommended for renal tissues, fibrin, connective tissues and
muscles
Mercuric Chloride
Permits brilliant staining of nuclear
and connective tissue fibers
Zenker’s fluid
Recommended mainly for tumor biopsies especially of the skin
Heidenhain’s Susa Solution
Commonly used for bone marrow
biopsies
B-5 fixative
Preserves lipids
Preserves mitochondria
Potassium Dichromate
Hardens tissues better and more rapidly than Orth’s fluid
Regaud’s Fluid or Muller’s Fluid
Demonstrates Rickettsiae and other bacteria
Orth’s fluid
Suitable for Aniline stains
Picric Acid
Does not need washing out
Bouin’s Solution
Recommended for fixation of
embryos and pituitary biopsies.
Bouin’s Solution
Suitable only for small pieces of tissues or
bones
trichloroacetic acid
Can irritate the eyes producing
conjunctivitis
osmium tetroxide
Permanently fixes fat
Flemming’s Solution
Recommended for the study of water diffusible
enzymes especially phosphatases and lipases
acetone
Highly explosive when dry
Picric Acid
