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

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: To prevent autolysis (self – destruction). Prevent putrefaction (bacterial attack). 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: Formaldehyde. Chromic Acid. Acetone. Ethyl Alcohol. Picric Acid. Acetic Acid. Mercuric Chloride. 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 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

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

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

Cytoplasm: Vaculation Granulation.

Some fixatives

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).

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.

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.

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.

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.