Dr. Waqas Nawaz PMAS arid agriculture university rawalpindi

1. WHAT IS FIXATION???????
GIVE THE DETAIL OF DIFFERENT FIXATIVES USE
FOR LIGHT MICROSCOPE
SUBMITTED BY:
SUBMITTED TO:
11-arid-959 M.UMAR
Dr. RIAZ HUSSAIN PASHA 11-arid-975 Waqas nawaz

2. Fixation
• The decisive step in obtaining a permanent
microscopic slide
• A physical or chemical process that stops as
soon as possible vital cellular processes,
maintaining with minimal alteration the
shape, volume, spatial and molecular relation
between elements

3. Fixation
• By fixation we intend:
1. To conserve the tissue from autolysis and
bacterial attack
2. To prevent the loss of cellular constituents
3. To increase optical differentiation of cellular
structures
4. To increase tissue consistency, in order to
facilitate their going through the other steps of
the technique – especially slicing

4. Fixatives classification
• Physical – heat, microwaves
• Chemical:
– Aldehydes – formaldehyde, glutaraldehyde,
acrolein
– Oxidizing agents – osmium tetraoxide,
potassium permanganate, potassium
dichromate
– Protein denaturating agents – acetic acid,
methyl alcohol, ethyl alcohol
– Miscellaneous – mercuric chloride, picric acid,
non aldehyde containing fixatives

5. Fixatives classification
• Mixtures of fixatives:
– By chemical nature:
• Aqueous
• Alcoholic
– By purpose:
• Universal / topographic
• Cytologic

6. Fixation mechanism
• Formes cross-links between proteins, thereby
forming a gel – keeps structures in their in vivo
relations to one another
• Soluble proteins are fixed to structural
proteins – insoluble → gives mechanical
strength for next steps
• Protein denaturation

7. Qualities of a good fixative
• Good tissue penetration
• Stabilizes the tissue, preserving the character
and distribution of cellular components
• Prevents fixation artefacts
• Prevents structure deformation – maintaining
shape and volume
• Preservs cellular constituents

8. Qualities of a good fixative
• Destroys microorganisms
• Extracts inactivated autolytic enzymes
• Increases tissue consistancy
• Confers optical differentiation
• Maintains its chemical composition
• Cheap, nontoxic, nonflamable, nonirritant

9. Post-fixation treatments
• Decalcification – for bone
• Sample dissociation
• Mordansation – helps both fixation and
staining

10. FIXATION
Samples should be fixed immediately after they are removed from the body.
Fixation (conservation) is used to:
- terminate cell metabolism
- prevent AUTOLYSIS (degradation of tissues by own enzymes)
- kill pathogenic microorganisms (prevent decomposition of tissue
by bacteria, fungi or viruses)
- harden tissue as a result of their cross-linking or denaturing
protein molecules
Requirements:
- rapid penetration of fixative into the tissue
- preservation of the structure
- maintenance of the affinity for dyes (stainability)
PHYSICAL FIXATION
Heat, microwave, freezing

11. CHEMICAL FIXATION
Submerging samples in the fluid fixative (immersion-fixation).
Perfusion by the fixative (perfusion-fixation in an experimental use).
Some fixatives promote cross-linking of proteins (formaldehyde, glutaraldehyde),
the other precipitation of proteins (picric acid, mercuric dichloride).

12. TYPES OF CHEMICAL FIXATIVES
FORMALDEHYDE fixatives
Formalin – trade name of 37 – 40% formaldehyde aqueous solution
10-20% neutral formalin (formic acid, which is present in the solution, is
neutralized by calcium carbonate), formalin saline (NaCl is added) buffered
formalin (phosphate buffer), Baker´s fluid (calcium chloride; for fixation of lipids)
Paraformaldehyde (histochemistry, electron microscopy)
Picric acid fixatives - fixation of glycogen (Bouin´s fluid)
Mercuric dichloride - containing fixatives: SUSA fluid, Zenker´s fluid
(black precipitates of mercury developed in tissue must be removed by
iodination)
Less used fixatives: methanol (smear), 96% ethanol (Nissl method)
cooled acetone (histochemistry)

13. Formaldehyde
Usually in form of
paraformaldeyde powder
or 37% to 16% aqueous
solution
•Low MW makes it one of the best penetrating of all the fixatives, thus it
is widely used in fixation of resistant materials, such as seeds, spores,
plant material, etc., usually in conjunction w/ another aldehyde.
•Formalin contains many impurities, so formaldehyde for use in EM is
normally prepared from the dissolution, heating, and alkalination of
powdered paraformaldehyde. Since this solution contains no inhibitors, it
has a shelf life of only a few weeks.

14. Glutaraldehyde
•Glutaric acid dialdehyde, a 5 Carbon dialdehyde, is the most widely applied fixative
in both scanning and transmission electron microscopy.
Most highly cross-linking of all the aldehydes. GTA fixation is irreversible.
•In TEM, buffered GTA has the reputation of providing the best ultrastructural
preservation in the widest variety of tissue types of any known chemical fixative.

15. Osmium Tetroxide (OsO4)
•A non-polar tetrahedral molecule with a
•molecular weight of 254 and solubility water
•and a variety of organic compounds.
•Its principle utility is its ability to stabilize and stain lipids- preferentially
unsaturated fatty acids
•Commercially available as a coarse yellow crystalline material packaged in glass
ampoules sealed under inert gas. Similarly packaged aqueous solutions are also
available.
•An additive, non-coagulative type of fixative,
but lacks the ability to crosslink many proteins.
•Very poor rate of penetration

16. Basic factors affecting chemical fixation
pH (Isoelectric point)
Total ionic strength of reagents
Osmolarity
Temperature
Length of fixation
Method of application of fixative