dr. samah kotb lecturer of biochemistry 2015 histology techniques cls 322
TRANSCRIPT
fixation
Dr. Samah KotbLecturer of Biochemistry
2015
Histology Techniques CLS 322
WHAT IS FIXATION?
Fixation is a process by which the internal constituents of
tissue are preserved.
The aim of fixation
• General aim: preservation of the shape,
structure, and chemical constituents of the tissue.
• Specific aim:
1) To prevent autolysis (self – destruction).
2) Prevent putrefaction (bacterial attack).
3) Harden of the tissue.
Principle of fixation
• 1. Involved denaturation or precipitation
(coagulation) of the protein in the tissue. Protein is
converted from the colloidal state (semi solid) to a
solid state (semi gel).
• 2. The cell membrane is killed and loses its property of
semi permeability within and it will be no longer able to
regulate the osmotic pressure in and out the cells.
• 3. The process also converts the tissue into an inert
spongy mass which makes it more rapidly permeable to
the stains.
classification of fixative
According to the action:
1. Additive. 2. Non – additive.
According to the content:
1. Simple. 2. Compound.
Latest classification:
1. Aldehydes. 2. Oxidizing.
3. Physical: heat 4. Miscellaneous: picric acid.
5. not oxidizing – not aldehyde.
Simple Fixative
• Made of one chemical substance.
• Example:
1. Formaldehyde.
2. Chromic Acid.
3. Acetone.
4. Ethyl Alcohol.
5. Picric Acid.
6. Acetic Acid.
7. Mercuric Chloride.
8. Osmic Acid.
• These fixatives fix by precipitating proteins and in this
case they are called protein precipitants. As Ethyl alcohol,
Acetic Acid & Potassium Dichromate at pH less than 3.7.
• Or by forming additive compounds (denaturing) and they
are called in this case non - protein precipitants. As
Formaldehyde, Osmic Acid & Potassium Dichromate at
pH more than 3.7.
Compound Fixatives
• Made of two or more of the simple fixatives.
Classified into:
• 1. Micro-anatomical fixatives
• 2. Cytological fixatives
• 3. Histochemical fixatives
• 4. Electron Microscope fixatives
1. Micro-anatomical fixatives: preserve and fix the various layers of
tissue and cells; to allow the study of their general structure. Ex:
10% formal saline, Zenkers’ solution, Bouins’ solution .
2. Cytological fixatives: preserve and fix the constituents
and elements of the cells. They divided into two groups:
i- Nuclear fixatives: used when we are interested in the
nuclear study. Ex: Carnoys’ fluid & Flemmings’ fluid.
ii- Cytoplasmic fixatives: used when we are studying the
Cytoplasmic elements. Ex: Hellys’ fluid, Flemmings’ fluid
without Acetic Acid.
3. Histochemical fixatives: used for the histochemical
investigations (ex: for enzymes). Ex: Cold Acetone, Cold
Absolute Alcohol & 10% buffered Formalin.
4. Electron Microscope fixatives: used when we are
dealing with specimens to be examined with E.M. Ex:
Osmic Tetroxide, Glutaraldehyde & acetaldehyde
Acrolein.
Other Techniques of Fixation
Vapour Fixation: are used when we want to avoid
liquid fixatives. That is possible by heating some
liquid fixatives to get their vapour. Ex:
1. Formaldehyde Vapour (heat at 50º – 80º C).
2. Acetaldehyde Vapour (heat at about 80ºC).
Freeze Drying: used to preserve tissue substances. Mainly
used in histochemistry.
Freeze substitution: this method is a substitute to freeze
drying. It does not need an apparatus like freeze drying. It is
run at low temperature in liquid dehydrating agents which
are also fixatives. First the tissue is quenched (initial rapid
freezing) and then transferred immediately to such fluid like
cold acetone or Rossmans’ fluid.
Factors affect fixation
Factors affect fixation:
1) pH.
2) Temperature.
3) Penetration of fixative.
4) Volume of tissue.
According to previous factors we can determine the
concentration of fixative and fixation time.
Effective of bad fixation
Nucleus:
1. Pyknosis.
2. Karryorhosis.
3. Karryolysis.
Cytoplasm:
1. Vaculation
2. Granulation.
Some fixatives
10% Formal Saline
To avoid formation of formic acid, Marble chips (Ca.
Carbonate) should be added to neutralize the solution.
Thin blocks 1.5 X 1.0 X 0.3 cm take about 24 hrs. to fix
(optimum fixation takes 7 days).
100 ml Formaldehyde (40%)
8.5 gm Sodium Chloride
900 ml Tape Water
10% Formal Calcium
Is widely used. The addition of Calcium Chloride
preserves phospholipids whereas the addition of
Calcium Acetate has the advantage of buffering
solution.
100 ml Formaldehyde (40%)20 gm Calcium Acetate or Calcium Chloride
to 1000 ml Tape Water
Zenkers’ Solution
Immediately before usage; add 5% Acetic Acid. In this case, the
solution is called Zenker – Acetic. Thin blocks fix for 3 – 18 hrs.
And if we add 5 % Formalin; the solution called Zenker – Formal
(Hellys’ fluid). Here, thin blocks fix for 6 – 24 hrs.
5 gm Mercuric Chloride2.5 gm Potassium Dichromate1 gm Sodium sulphate (optional)
100 ml DW
Bouins’ Solution
Thin blocks are fixed for 6 – 24 hrs and then transferred to 70%
alcohol. The yellow color of the picric acid is an advantage with
very small biopsies; and it should be removed from the sections
before staining by alcohol followed by 2.5 % Sodium
Thiosulphate.
75 ml Saturated Picric Acid
25 ml Formalin (formaldehyde 40%)
5 ml Acetic Acid
Carnoys’ Solution
Generally tissues contain some fat which slows down the
penetration of fluids. The chloroform dissolves the fat and
allows the penetration of this fixative. Block of 3 mm thick fix in
30 – 90 minutes.
60 ml Absolute Alcohol30 ml Chloroform10 ml Acetic Acid