Fixation Fixation- it is series of complex chemical reactions between the fixative and the cell/ tissue components.

It produces stable and reproducible state. The ideal fixative will preserve tissues as in life like state as possible. This is never be completely achieved as metabolic processes of dying cell will cause changes. AIMS OF FIXATION. 1/ To deliberately and irreversibly alter the components of the living tissue, by rendering them insoluble in the various reagents which will subsequently be used ( processing and staining fluids). 2/To preserve the cell by preventing autolysis and putrefaction. Autolysis. = self digestion of cellular components by enzymes present in the cell. Putrefaction. = changes occurring as a result of bacterial enzymes. 3/ To enable the subsequent staining reactions, by enhancing the optical differences between cells and structures. 4/To prevent the distortion by physical forces and shrinkage. 5/To render the tissue resistant to changes caused by processing methods, e.g. hardening and shrinkage. Autolysis and Putrefaction. - These are the changes first seen on inadequately fixed tissue. - They proceed quickest at 37oC and are slowed down at 4oC. They are completely stopped by heat. Recognition of autolysis and putrefaction. -Cells tend to become pyknotic. They look small and dense. -Normal cells are = Vesicular, the chromatin is visible. or = pyknotic, the chromatin is dense. - Nuclei may also show karyorrhexis where the nuclei brake up into lumps. - May also see karyolysis where chromosome material dissolves giving a ghost nuclei. - Cytoplasm becomes excessively granular, swollen and looses its cytoplasmic contents. - Epithelia tends to desquamate ( cells come away from the basement membrane. Fixation: stabilises proteins quickly, doe not fix everything-lipids are preserved not fixed. A fixative do everything a preservative does but also increase optical differences between tissue components and make tissue resistant to the subsequent processing. TYPES OF FIXATIVE These can be separated into two rough groups; 1/ Those which stabilise proteins by precipitation. e.g. Ethyl alcohol, acetic acid, chromium tetroxide and picric acid.

2/ Those which stabilise proteins by other means (non precipitating agents). Those which bind proteins together by other means. e.g.Formalin, mercuric chloride, osmium tetroxide, gluteraldehyde and potassium dichromate. MECHANISMS OF FIXATION. 1/ By an aldehyde fixative. These are thought to work by cross linking between the aldehyde (e.g. formaldehyde) and basic lysine (amino acids) residues on the exterior of the protein. This will produce a bridge called either a lysine bridge or methylene bridge. The lysine residues on the inside of the protein do not react to begin with. Because of this the bridge formation or fixation is reversible in the first twenty four hours. After this the bridges both internal and external are produced and the fixation is irreversible. This reaction works best at a high pH of 9, and also works well at pH 7-7.4 the physiological pH of the body. Gluteraldehyde, a fixative used in electron microscopy, produces rapid and irreversible bridge formation. 2/ Other reactions of fixatives are less well known. e.g. mercuric chloride is thought to react with a number of amino acids including thiol, amino and hydroxyl groups. 3/ Some fixatives are oxidising agents,e.g. osmium tetroxide, another fixative of electron microscopy. The mechanism is not well known. IMPORTANT POINTS OF FIXATION. - usually not single fixing agents, but mixtures of fixing agents - - They must kill the cell quickly with little shrinkage, swelling or distortion. - Their rate of penetration effects its fixing ability. It must penetrate quickly. - As most fixatives penetrate they harden tissues and form a barrier to their own penetration. - Penetration is dependent on the tissue type. Denser tissues take longer to fix than cellular tissues. - It must render insoluble the substance of the cell and give good optical differentiation. - It must inhibit bacterial decay and autolysis - The end result of fixation is an artefact. The tissue is not in the same state as it was in life. The artifacts are controlled and we therefore now what to expect. Artifacts;- cell shrinkage. - cytoplasm turns from a SOL TO GEL. - chromatin is often precipitated. - cytoplasmic contents are often precipitated. - lipid disappears. - artifactual spaces develop in the tissue. - As much fixative solution as possible should be used. Around 20x the volume of the tissue. - The tissue should be removed from the body and placed in fixative as quickly as possible and left to fix for as long as possible. - The correct fixative for both the tissue and the stain must be chosen.

- After initial fixation the fixative should be changed. FACTORS INVOLVED IN FIXATION pH, temperature, penetration, osmolarity, concentration, duration COMMON FIXATIVES Formalin. - Very practical. - Is a 36-40% solution. - Made from the oxidation of methyl alcohol. - Standard fixative strength 10% formalin (4% formaldehyde). - Formic acid will form from formalin over time. This is overcome by buffering the solution with phosphate or acetate salts. - Formalin is made up in tap water as the pH is more suitable (distilled water is acid in nature). Types of formalin based fixatives. 1/ Formal saline. - this is an isotonic solution, made up in physiological saline. It decreases shrinkage or swelling of the tissue. 2/Formal calcium. - 10% formalin plus 1% calcium chloride. This is useful for lipid work. 3/Formal ammonium bromide. - Good for CNS work, especially for gold impregnation methods. 4/Formal alcohol. - 10% formalin made up in 70% alcohol. Is a very rapid fixative and good for preservation of glycogen. Mercury based fixitives Picric acid based fixitives Potassium dichromate fixitives Heidenhains susa Microwave fixation- fixation by heat. Microwave energy interacts with dipolar molecules, causing oscillation at 2450 MHz. Water molecules and protein polar side chains have their thermal energy increased so heating the tissue. The optimum temp for fixation of tissues is 45-55oC. Under heating causes poor sectioning whereas overheating causes vacuolation, over stained cytoplasm and pyknotic nuclei. Rapid fixation for rapid diagnosis. Fixation is not required for frozen sections, histochemistry, immunifluorescence