1. Fixation
Is the fundamental step in tissue processing. It has to be complete and adequate
The principle of fixation:
The principle involved is:• the denaturation or• precipitation (coagulation) of the protein in the tissue
The process converts the tissue protein from colloidal (semi sol) in to a solid (semi gel) state
Precipitation involves the appearance of mixture of granular or reticular solids suspended in a fluid. Coagulative fixative ( chromic, acetone) transforms the protoplasm of the cell into a network of reticulum which will allow:
The embedding wax to pass throw the tissue
Good quality of sectioning

2. The process converts the tissue into inert spongy mass, which makes it more rapidly permeable to stains
Fixation takes place from the outer surface of the specimen towards its center
Cell membrane is killed and looses its property of semi permeability, it will be no longer able to regulate osmotic gradient within and outside the cell
Fixative should provide more stable bonds, which will hold tissue molecules together during dehydration and subsequent treatment

3. Protect the tissue from disruption during embedding and sectioning
The aim of fixation:
The aim of fixation is the preservation of shape, structure, relationship and chemical constituents of the tissue (physically and chemically) in as life-like manner as possible.
To preserve the relationship between tissues components with minimum alteration from the living state
Protect the tissue from disruption during embedding and sectioning
Prepare the tissue for subsequent treatment (staining)
Fixation is of selective rather than of general effectiveness, to study lipid, one would select osmium tetroxide as a fixative and avoid potassium permanganate

4. The criteria an ideal fixative:
Works quickly with minimum distortion, penetrate the tissue rapidly, and evenly
It should have no hypotonic or hypertonic effect on tissue (causes minimum shrinkage or swelling)
It should coagulates the tissue proteins (e.g. protoplasmic material) to prevents the diffusion of tissue substances
Inhibit putrefaction and autolysis of tissue
Induce tissue hardening to facilitate cutting
Protect the tissue from harmful effect of subsequent steps (e.g. dehydration)
Mordanting (enhance staining method)
Preserve the color (no color change), especially in the museum
It should Not produce artifacts
not harmful to the user

5. The criteria an ideal fixative…..
12. Not harmful to containers 13. It should not need treatment after fixation 14. Easy to make and not expensive

6. Types of fixatives: 1- Simple fixatives 2-Compound fixatives
contain one chemical substance which has its own particular effect or action on one or more of the tissue substances or constituents
Consists of two or more of the simple fixatives

7. Non-protein precipitate (denaturing)
1- Simple fixatives
Protein precipitate
Non-protein precipitate (denaturing)
Mercuric chloride
Chromic acid
Acetic acid
Ethyl alcohol
K cl (less than 3.7 pH)
Formaldehyde
Osmium tetroxide
K cl (less than 3.7 pH)

8. 2-Compound fixatives Microanatomical: B) Cytological:
10% formal-saline
Zenker’s solution
Bouin’s solution
heidenhain’s solution
B) Cytological:
Nuclear
Flemmings' fluid
Carnoy’s fluid
ii) Cytoplasmic
Flemmings' fluid W/O acetic acid
Hellys fluid
Formalin fixation with post-chroming

9. Hints:
Bouin and 10% formalin are only microanatomical fixative
Carnoy is only nuclear fixative
Zenker is microanatomical and nuclear fixative
Helly is microanatomical and cytological (nuclear and cytoplasmic)
10% buffered formalin and formal sublimate are the same
Histochemical fixataves is different from all these types

10. Histochemical fixatives: Electron microscope fixatives:
Used for histochemical investigations (e,g. for enzymes
1o% buffered formalin
Cold acetone
Cold absolute alcohol
Electron microscope fixatives:
Glutaraldehyde
Acetaldehydes acrolein
Osmic tetroxide
Vapour fixatives:
Used when wants to avoid liquid fixatives
Fixation is possible by heating some liquid fixatives to get their vapour
Formaldehyde vapour-50-80
Acetaldehyde vapour about 80
Glutaraldehyde vapour (50% aqueous)-60-80