Is the art and science performed by histotechnologists to produce a tissue section of good quality that will enable the pathologist to diagnose the presence/absence of disease

Preserve the morphologic and chemical intergrity of the cell

To harden and protect the tissue from the trauma of further handling

Is a form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5-3% potassium dichromate for 24 hours to act as mordant

Is the process of removing excess fixative from the tissue after fixation in order to improve staining and remove artifacts from the tissues

Used to remove chromates, formalin, and osmic acid

Used to wash out excess amount of picric acid

Used to remove excess mercuric fixative

The chemical constituent of the fixative is taken in and becomes part of the tissue; stability by forming cross-links

The fixative is not incorporated into the tissue; stability by removing the bound water attached to H-bonds of certain groups within the protein molecule

Main factors involved in fixation

Hydrogen ion concentration pH

Surgical specimens temperature

Electron microscopy & some histochemistry temperature

Bacteriology and blood films temperature

For urgent biopsies temperature

For tissues with tuberculosis temperature

Electron microscopy thickness of fixation

Light microscopy thickness of fixation

Isotonic (340 mOsm) & hypotonic

Hypertonic

Slightly hypertonic solutions (400-450 mOsm)

Permits the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question

Preserve the specific parts and particular microscopic elements of the cell itself

Preserve the nuclear structures; usually contains glacial acetic acid as primary component (pH 4.6 or less)

Preserve cytoplasmic structures; must never contain glacial acetic acid (pH of more than 4.6)

Preserve the chemical constituents of cells and tissues

Commercially available solution of formaldehyde (gas produced by the oxidation of methyl alcohol) : 4% formaldehyde diluted 1:10 or 1:20 to make 10% or 5% solution

Unsatisfactory for routine fixation

Formaldehyde (formalin) fixation time

A simple microanatomical fixative made up of saturated formaldehyde diluted to 10% with sodium chloride; recommended for central nervous tissues and general post-mortem tissues. Advantage : ideal for most staining techniques - Disadvantages : a.) slow fixative, b.) metachromatic reaction of amyloid is reduced

Recommended for preservation and storage of surgical, post-mortem and research specimens ▪ Advantage : best fixative for tissues containing iron pigments and elastic fibers ▪ Disadvantage : time-consuming to prepare

Formol-mercuric chloride solution ▪ Recommended for routine post-mortem tissues ▪ Advantage : no need for “washing out” ▪ Disadvantages : a.) forms mercuric chloride deposits, b.) does not allow frozen sections to be made, c.) inhibits the determination of the extent of tissue decalcification

Advantages : a.) can be used for rapid diagnosis, b.) good for preservation of glycogen and for micro-incineration technique, c.) used to fix sputum ▪ Disadvantage : causes partial RBC hemolysis

Made up of two formaldehyde residues linked by three carbon chains; recommended for enzyme histochemistry and electron microscopy ▪ Advantages : a.) preserves plasma proteins better, b.) more pleasant & less irritating to the nose, & doesn’t cause dermatitis ▪ Disadvantages : a.) more expensive & less stable, b.) tends to make tissues more brittle

Glutaraldehyde solution that is used for small tissue fragments and needle biopsie

Most common metallic fixative ▪ widely used as a secondary fixative reacting with a number of amino acid residues and accompanied by spectroscopic changes, probably due to reacion with histidine residues ▪ routine fixative of choice for preservation of cell detail in tissue photography ▪ recommended for renal tissues, fibrin, connective tissues & muscles

Used in 4% aqueous solution of basic lead acetate ▪ recommended for acid mucopolysaccharides ▪ fixes connective tissue mucin ▪ Disadvantage : it takes up carbon dioxide to form insoluble lead carbonate (remedy: filtration or by adding acetic acid drop by drop)

To remove mercury deposits: (treating the section with 0.5% iodine solution in 70% ethanol for 5-10 minutes before staining)

Mercuric chloride stock solution with glacial acetic acid (added before its use to prevent turbidity and formation of a dark precipitate) ▪ a good general fixative ▪ gives excellent staining results ▪ recommended for fixing small pieces of liver, spleen, connective tissue fibers & nuclei ▪ recommended for TRICHOME staining

Recommended mainly for tumor biopsies especially of the skin ▪ excellent cytologic fixative ▪ Advantages : a.) susa-fixed tissues may be transferred directly to 95% alcohol or absolute alcohol, b.) permits easier sectioning of large blocks of fibrous connective tissues ▪ Disadvantages : a.) RBC preservation is poor, b.) Weigert’s method of staining elastic fibers is not possible

Excellent microanatomic fixative for pituitary gland, bone marrow and blood-containing organs (spleen, liver) ▪ Advantage : nuclear fixation and staining is better than Zenker’s ▪ Disadvantages : similar to Zenker’s except that brown pigments are produced if tissues are allowed to stay in the fixative for more than 24 hours due to RBC lysis (remedy: immerse the tissue in saturated alcoholic picric acid or sodium hydroxide)

Commonly used for bone marrow biopsies ▪ Advantage : rapid fixation can be achieved in 1/2 -2 hours ▪ Disadvantage : overfixation hardens the tissues and makes cutting difficult

Used in 1-2% aqueous solution ▪ precipitates all proteins ▪ adequately preserves carbohydrates ▪ a strong oxidizing agent

Used in a 3% aqueous solution ▪ preserves lipids and mitochondria

Recommended for demonstration of chromatin, mitochondria, mitotic figures, golgi bodies, RBC and colloid-containing tissues ▪ Advantage : hardens tissues better and more rapidly than Orth’s fluid ▪ Disadvantages : a.) produce precipitates of sub-oxide, b.) does not preserve fats, c.) prolonged fixation blackens tissue pigments (remedy: washing the tissues in running tap water prior to dehydration)

Recommended for study of degenerative processes and tissue necrosis ▪ demonstrates rickettsiae and other bacteria ▪ Disadvantage : same as in Regaud’s fluid

Dyes the tissues (yellow color removed by treatment with another acid dye or lithium carbonate or washing with changes of 50% and 70% ethanol) ▪ excellent fixative for glycogen demonstration ▪ suitable for Aniline stains ▪ Advantages : a.) precipitates all proteins, b.) stable ▪ Disadvantages : a.) not suitable for frozen sections, b.) will produce excessive staining of tissues, c.) highly explosive when ddry

Solidifies at 17℃ ▪ an essential constituent of most compound nuclear fixatives ▪ Advantage : fixes and precipitates nucleoproteins, chromosomes, and chromatin materials ▪ Disadvantage : contraindicated for cytoplasmic fixation

Rapidly denatures and precipitates proteins ▪ must be used in concentrations ranging 70% to 100% ▪ ideal for small tissue fragments ▪ excellent for glycogen preservation ▪ Advantage : may be used as a fixative and dehydrating agent ▪ Disadvantages : a.) dissolves fats and lipids, b.) causes glycogen granules to move towards the poles or ends of the cells (polarization)

A pale yellow powder which dissolves in water to form a strong oxidizing solution ▪ fixes conjugated-fats and lipids permanently ▪ used extensively for neurological tissues (fixes myelin and peripheral nerves well)

Recommended for fixation of embryos and pituitary biopsies ▪ excellent fixative for preserving soft and delicate structures ▪ preferred fixative for tissues to be stained by Masson’s trichrome for collagen, elastic or connective tissue

Excellent fixative for glycogen ▪ Advantage : better and less messy than Bouin’s solution

Excellent for fixing dry and wet smears, blood smears and bone marrow tissues ▪ Advantage : fixes and dehydrates at the same time ▪ Disadvantage : if left for more than 48 hours, tissues may be overhardened and difficult to cut

Used for fixing touch preparations

Used at concentrations of 70-100% ▪ may be used as a simple fixative ▪ fixes blood, tissue films and smears; preserves nucleoproteins and nucleic acids ▪ Disadvantages : a.) causes polarization, b.) strong reducing agent

Recommended for fixing chromosomes, lymph glands, and urgent biopsies ▪ used to fix brain tissue for the diagnosis of rabies considered to be the most rapid fixative ▪ Advantages : a.) preserves Nissl granules and cytoplasmic granules, b.) suitable for small tissue fragments ▪ Disadvantages : a.) suitable only for small pieces of tissues, b.) leads to polarization

Recommended for fixing mucopolysaccharides and nuclear proteins

Most common chrome-osmium acetic acid fixative used ▪ excellent fixative for nuclear structures ▪ Advantages : a.) permanently fixes fat, b.) relatively less amount of solution is required for fixation ▪ Disadvantages : a.) solution deteriorates rapidly, b.) depress staining power of hematoxylin, c.) very expensive

Made up of only chromic and osmic acid ▪ recommended for cytoplasmic structures particularly the mitochondria

Advantages : a.) may be used as a weak decalcifying agent, b.) softening effect on dense tissues ▪ Disadvantage : poor penetrating agent

Used at cold temperature (-5℃ to 4℃) ▪ recommended for the study of water diffusible enzymes (phosphatases and lipases) ▪ used in fixing brain tissues for diagnosis of rabies ▪ Advantage : used as a solvent forr certain metallic salts to be used in freeze substitution techniques for tissue blocks ▪ Disadvantage : evaporates rapidly

Involves thermal coagulation of tissue proteins for rapid diagnosis (frozen tissue sections & bacteriologic smears) ▪ Advantages : a.) fixation is better, b.) preserves nuclear and cytoplasmic details, c.) suitable for frozen tissue preparation ▪ Disadvantages : a.) destroys RBC, b.) dissolves starch and glycogen

Works as a physical agent similar in mechanism to vacuum, oven, and agitation to increase the movement of molecules and accelerate fixation

Commonly used in pathology for the demonstration of various antibodies

A.) preserve the maximum enzyme activity at its original localization, B.) preserve structural integrity : fixed in 4% formaldehyde or formol saline overnight : acetone or formaldehyde and washed in distilled water prior staining (fresh frozen sections

The whole procedure performed at 4℃ fixatives: osmium tetroxide, glutaraldehyde, paraformaldehyde

For Microwave technique, the oscillation must be at a frequency of

Produced under acid conditions - fixation in phenol-formalin or neutral buffered formalin

Due to partial coagulation of partially fixed protein by ethanol or by incomplete wax impregnation during subsequent histological processing - may be found in surgical specimens particularly in liver biopsies