1. Lecturer of Biochemistry
fixation
Histology Techniques
CLS 322
Dr. Samah Kotb
Lecturer of Biochemistry
2015

2. WHAT is fixation?
Fixation is a process by which the internal constituents of tissue are preserved.

3. The aim of fixation
General aim: preservation of the shape, structure, and chemical constituents of the tissue.
Specific aim:
To prevent autolysis (self – destruction).
Prevent putrefaction (bacterial attack).
Harden of the tissue.

4. 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).

5. 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.

6. classification of fixative
According to the action:
1. Additive Non – additive.
According to the content:
1. Simple Compound.
Latest classification:
1. Aldehydes Oxidizing.
3. Physical: heat Miscellaneous: picric acid.
5. not oxidizing – not aldehyde.

7. Simple Fixative Made of one chemical substance. Example: Formaldehyde.
Chromic Acid.
Acetone.
Ethyl Alcohol.
Picric Acid.
Acetic Acid.
Mercuric Chloride.
Osmic Acid.

8. 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.

9. Compound Fixatives 1. Micro-anatomical 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

10. 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 .

11. 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.

12. 3. Histochemical fixatives: used for the histochemical investigations (ex: for enzymes). Ex: Cold Acetone, Cold Absolute Alcohol & 10% buffered Formalin.

13. 4. Electron Microscope fixatives: used when we are dealing with specimens to be examined with E.M. Ex: Osmic Tetroxide, Glutaraldehyde & acetaldehyde Acrolein.

14. 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).

15. Freeze Drying: used to preserve tissue substances
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.

16. Factors affect fixation

17. Factors affect fixation:
pH.
Temperature.
Penetration of fixative.
Volume of tissue.
According to previous factors we can determine the
concentration of fixative and fixation time.

18. Effective of bad fixation
Nucleus:
Pyknosis.
Karryorhosis.
Karryolysis.

20. Cytoplasm:
Vaculation
Granulation.

21. Some fixatives

22. 10% Formal Saline
100 ml
Formaldehyde (40%)
8.5 gm
Sodium Chloride
900 ml
Tape Water
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).

23. Calcium Acetate or Calcium Chloride
10% Formal Calcium
100 ml
Formaldehyde (40%)
20 gm
Calcium Acetate or Calcium Chloride
to 1000 ml
Tape Water
Is widely used. The addition of Calcium Chloride preserves phospholipids whereas the addition of Calcium Acetate has the advantage of buffering solution.

24. Sodium sulphate (optional)
Zenkers’ Solution
5 gm
Mercuric Chloride
2.5 gm
Potassium Dichromate
1 gm
Sodium sulphate (optional)
100 ml DW
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.

25. Formalin (formaldehyde 40%)
Bouins’ Solution
75 ml
Saturated Picric Acid
25 ml
Formalin (formaldehyde 40%)
5 ml
Acetic Acid
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.

26. Carnoys’ Solution
60 ml
Absolute Alcohol
30 ml
Chloroform
10 ml
Acetic Acid
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.