Histopathologic Techniques: Gregoris - Chapter 3: Fixation and Fixatives

Fixation

The first and most critical step in histotechnology

Fixation

Involves fixing and preserving fresh tissue for examination

putrefaction

decomposition of proteins by the action of enzymes from anaerobic bacteria

pH 6 and 8

This is the satisfactory hydrogen ion concentration for fixation

25 C

This is the traditional laboratory temperature for fixation of surgical spx

0-4 C

This is the ideal temperature for fixation in electron microscopy and some histochemistry

60 C

This is the temperature formalin is heated to for rapid fixation of urgent biopsy spx

100 C

Th

This is the temperature formalin is heated to for fixing tuberculosis tissue

1-2 mm2

Ideal size of tissue block for electron microscopy

2 cm2

Ideal size of tissue block for light microscopy

400-450 mOsm

What osmolality do best fixation results happen

Simple Fixatives

These are fixatives made up of only one component substance

Compound fixatives

These are fixatives that are made up of two or more fixatives

Microanatomical Fixatives

These are fixatives that permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship

Cytological fixatives

These are fixatives that preserve specific parts and particular microscopic elements of the cell itself

Nuclear fixatives

These are fixatives with glacial acetic acid that preserve the nuclear structures

Cytoplasmic fixatives

These are fixatives with glacial acetic acid that preserve cytoplasmic structures

Ethanol, methanol, Carnoy's Solution

These are the commonly used fixatives for nucleic acids

Histochemical Fixatives

These are fixatives that preserve the chemical constituents of cells and tissues

Baker's formol-calcium

Preserves phospholipids

Alcoholic Fixatives

Recommended for glycogen fixation in general

Alcoholic formaldehyde

Better fixative in human skin compared with neutral buffered formaldehyde

Neutral buffered formol saline

fixative for amino acid histochemistry

Fomaldehyde vapor

fixative for amino acid histochemistry along with neutral buffered formol saline

Rossman's fluid

Best fixative for glycogen retention and preservation

Cold Absolute alcohol

Best fixative for glycogen retention and preservation besides Rossman's Fluid

Karnovsky's paraformaldehyde-glutaraldehyde

This is the double aldehyde fixative mixture particularly useful for electron cytochemistry

Acrolein

A two mixture fixative of glutaraldehyde and formaldehyde that may be useful for immersion fixation of surgical biopsies

Aldehyde fixatives

These are satisfactory for routine paraffin section

4%

Concentration of formalin commonly used

10%

Concentration of formalin commonly used for tissue fixation

Formalin

Fixative recommended for colored tissue photography

10% Formol saline

This is a simple microanatomical fixative made up of saturated formaldehyde (40% weight volume) diluted to 10% with sodium chloride

10% Formol saline

This fixative is recommended for central nervous tissue and general post-mortem tissue for histochemical examinations

10% neutral buffered formalin

This is recommended for preservation and storage of surgical, post-mortem, and research specimens

Phosphate-buffered fomalin

This is the other name of 10% neutral buffered formalin

10% neutral buffered formalin

This is the best fixative for tissues containing iron pigments for elastic fibers which do not stain well after Susa, Zenker, or Chromate fixation

Formol corrosive

This is recommended for routine post-mortem tissues

formol-mercuric chloride solution

This is the chemical name of Formal corrosive

Formol corrosive

recommended for Neutral fats, Phospholipids, lipids

Formol corrosive

This is excellent for many staining procedures including silver reticulum methods

Alcoholic Formalin (Gendre's fixative)

This is good for the preservation of glycogen and for micro-incineration technique

Alcoholic Formalin (Gendre's fixative)

This is used to fix sputum, since it coagulates mucus

Glutaraldehyde

This fixative is made of two formaldehyde residues, linked by three carbon chains

Glutaraldehyde

This acts in a similar manner to formaldehyde and is sometimes utilized for routine light microscopy

Mercuric chloride

This is the most common metallic fixative

Saturated iodine solution in 96% alcohol

Added to mercuric chloride for the removal of black mercurial deposits

Mercuric chloride

This is widely used as a secondary fixative reacting with a number of amino acid residues and accompanied by spectroscopic changes

Mercuric chloride

This fixative has greater affinity to acid dyes and is preferred in lieu of formaldehyde for cytoplasmic staining

Mercuric chloride

This is the routine fixative of choice for preservation of cell detail in tissue photography

Mercuric chloride

This fixative is recommend for renal tissues, fibrin, connective tissues, and muscles

Mercuric chloride

This fixative causes considerable lysis of red blood cells and removes much iron from hemosiderin

Zenker's Fluid

This solution is formed by adding glacial acetic acid before use of mercuric chloride

Zenker-formol (Helly's solution)

This solution is formed by adding strong formaldehyde before use of mercuric chloride

Zenker's Fluid

This fixative is recommended for fixing small pieces of liver, spleen, connective tissues fibers, and nuclei

Zenker's Fluid

This fixative is not stable after the addition of acetic acid

Zenker's Fluid

This fixative does not permit cutting of frozen sections

Zenker's Fluid

This fixative must be washed in running water for several hours before processing

Zenker-formol (Helly's solution)\

This is an excellent microanatomic fixative for pituitary gland, bone marrow, and blood containing organs such as spleen and liver

Heidenhain's Susa Solution

Recommended mainly for tumor biopsies especially of the skin; it is an excellent cytologic fixative

Heidenhain's Susa Solution

This fixative does not allow Weigert's method of staining elastic fibers

96-100% alcohol

This is the solution tissue is directly transferred after using Heidenhain's Susa Solution

B5 fixative

This fixative is commonly used for bone marrow biopsies

Chromic acid

This is used in 1-2% aqueous solution, usually as a constituent of a compound fixative

Chromic acid

This chromate fixative precipitates all proteins and adequately preserves carbohydrates

Potassium Dichromate

This chromate fixative preserves lipids and mitochondria

Regard's (Muller's) Fluid

This chromate fixative is recommended for demonstration of chromatin, mitochondria, mitotic figures, golgi bodies, RBC and colloid-containing tissues

Orth's Fluid

Recommended for study of early degenerative processes and tissue necrosis

Orth's Fluid

This chromate fixative preserves myelin better than buffered formalin

Lead fixative

These fixatives are used in 4% aqueous solutions of basic lead acetate

Lead fixative

This is recommended for acid mucopolysaccharides

Picric acid

This is normally used in strong aqueous solution (1%)

Picric Acid

This is an excellent fixative for glycogen demonstration

Bouin's Solution

recommended for fixation of embryos and pituitary biopsies

Brasil's alcoholic picroformol fixative

Excellent Picric acid fixative for glycogen

Acetic acid

This is normally used in conjunction with other fixatives to form a compound solution

17 C

Temperature where glacial acetic acid solidifies

Absolute alcohol

This fixative can be used to fix and preserve glycogen, pigments, blood, tissue films and smear

Methyl acohol 100% / Methanol

This fixative is excellent for fixing dry and wet smears, blood smear, and bone marrow tissues

isospropyl alcohol 95%

This is used for fixing touch preparations

Ethyl alchohol

This alcohol fixative is frequently incorporated into compound fixatives for better results

Carnoy's Fluid

This is recommend for fixing chromosomes, lymph glands, and urgent biopsies

Newcomer's Fluid

This is an alcohol fixative recommended for fixing mucopolysaccharides and nuclear proteins

Osmium Tetraoxide

This is a pale yellow powder which dissolves in water

Flemming's solution

This is the most common chrome-osmium acetic acid fixative used

Flemming's solution

This is recommended for nuclear preparation of sections

Flemming's solution without acetic acid

Recommended for cytoplasmic structures particularly mitochondria

Tricholoroacetic acid

This fixative has its marked swelling effect on tissues serves to counteract shrinkage produced by other fixatives.

Acetone

Used in fixing brain tissues for diagnosis of rabies aside from Carnoy’s fluid

Heat Fixation

This procedure involves thermal coagulation of tissue proteins for rapid diagnosis, usually employed for frozen tissue sections and preparations of bacteriologic smears

Secondary Fixation

This is the process of placing and already fixed tissue in a second fixative

Post-chromatization

This is a form of secondary fixation whereby a primarily fixed tissue is place in aqueous solution

Washing Out

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

Removes Artifact chromate

Acid alcohol or Tap water

Remove artifact from osmic acid

Tap water

Removes Malarial pigments

Saturated alcoholic picric acid

Remove picrates

70% alcohol