Fixation

Dr. Waqas Nawaz
PMAS arid agriculture university rawalpindi

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

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: To conserve the tissue from autolysis and bacterial attack To prevent the loss of cellular constituents To increase optical differentiation of cellular structures 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 (OsO 4 ) 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