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

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