Fixation

Flashcards covering key vocabulary and concepts from the lecture notes on fixation techniques.

Numbering

Entering the details of the specimen in a log book. Classification includes surgical, autopsy, or cytologic specimen.

Fixation

The killing, penetration and hardening of tissues to preserve cells as close to their original state as possible.

Heat Fixation

A physical fixation method used mainly in microbiology for bacterial smears, and occasionally for frozen specimens in histopathology.

Microwave Technique

A physical fixation method that accelerates fixation, staining, decalcification, immunohistochemistry, and EM by increasing molecular movement.

Temperature in Fixation

Room temperature for manual processing, 0-4°C for EM & Histochemistry, 40°C for Auto Technicon, 60°C for rapid biopsies (Formalin heated), and 100°C for tissues with TB (Formalin heated).

Thickness/Size for Fixation

1-2 mm² for Electron Microscopy, 2 cm² for Light Microscopy, and 4-5 mm thick for tissues (2 cm for lungs).

pH in Fixation

Use a fixative with a pH between 6-8.

Osmolality in Fixation

Slightly hypertonic, use isotonic solution. Hypertonic solutions may cause cell shrinkage, hypotonic solutions may cause cell swelling.

Concentration in Fixation

10% Formalin, 3% glutaraldehyde, 0.25% glutaraldehyde for immune EM.

Ideal Fixation Time

20-30 minutes following interruption of blood supply or removal from the body.

Factors Retarding Fixation

Large tissues, presence of mucus, blood, fat and cold temperature.

Factors Accelerating Fixation

Smaller & thinner tissues, agitation, heat (acceptable temperature is 37-56°C).

Volume of Fixative

20x the volume of specimen (20:1 ratio). For Osmium tetroxide: 5-10x the volume. For Museum preparations: 50-100x the volume.

Formalin Diffusion Rate

Approximately 1 mm/hour.

Additive Fixatives

Combine with or are absorbed by the tissue, stabilizing tissue proteins. Examples: Formaldehyde, Mercuric chloride, Chromium trioxide, Picric acid, Glutaraldehyde, Osmium tetroxide, Zinc sulfate or chloride.

Non-Additive Fixatives

Not absorbed by the tissue but alter tissue components. Examples: Acetone and Alcohol.

Microanatomical Fixatives

Preserve general microscopic tissue structures without altering intercellular relationships. Examples: 10% formol saline, 10% neutral buffered formalin, Heidenhain’s Susa, Zenker’s, Bouin’s, Brasil’s

Nuclear Fixatives

Preserve parts of the nucleus; usually contain glacial acetic acid and have a pH less than 4.6.

Cytoplasmic Fixatives

Preserve parts of the cytoplasm; usually do not contain glacial acetic acid and have a pH more than 4.6.

Histochemical Fixatives

Preserve chemical components of tissues, such as enzymes. Examples: 10% formol saline, Absolute ethyl alcohol, Acetone, Newcomer’s.

Formaldehyde/Formalin

37-40% aka 100% formalin. A common aldehyde fixative, penetration rate is 1 mm/hour.

10% Formol Saline

Diluted with distilled water and sodium chloride. It is classified as histochemical fixative, general post mortem and CNS tissue.

10% Neutral Buffered Formalin

Recommended for fixing tissue with iron pigments and elastic fibers.

Formol Corrosive

Contains formaldehyde and mercuric chloride. Recommended for lipids, neutral fats and phospholipids.

Glutaraldehyde

Recommended for enzyme histochemistry and Electron Microscopy (2.5% for small fragments, 4% <4mm thick).

Glyoxal

Fast acting fixative; surgical specimens fixed in 4-6 hours, small biopsy specimens in 45 minutes.

Mercuric Chloride

Most common metallic fixative. May form black mercury deposits, treat with alcoholic iodine.

Zenker’s Fluid

Contains mercuric fluoride and glacial acetic acid, recommended for fixing liver, spleen, connective tissue fibers and nuclei.

Heidenhain’s Susa

With TCA, Glac HAc & formalin, recommended for preserving tumor skin biopsies.

Chromic Acid (1-2%)

Recommended for preserving carbohydrates.

Potassium Dichromate (3%)

Preserves lipids and mitochondria.

Lead Fixatives

For acid mucopolysaccharide and tissue mucin.

Picric Acid

Can act as a fixative, stain and decalcifying agent. Excellent for Glycogen demonstration, imparts a yellow color.

Bouin’s Solution

Recommended for fixation of embryos, pituitary biopsies and endometrial curettings. NOT for kidneys & abolishes feulgen’s reaction.

Glacial Acetic Acid

Not used as routine fixative; compound fixative, recommended for nucleoproteins, solidifies at 17°C & contraindicated for cytoplasmic fixation.

Acetone

Can fixed and also dehydrate tissues at the same time. Preservation of enzymes such as lipases and phosphatases & for fixing brain tissues for the diagnosis of rabies. Use at ice cold temperatures negative 5-4°C.

Alcohol Fixatives

Rapidly denatures and precipitates proteins. Can act both as fixative and dehydrative agents. Ideal for small tissue fragments.

Carnoy’s Fixative

Most rapid fixative, recommended for fixing chromosomes and lymph glands. It contains absolute alcohol, glacial HAC and chloroform.

Osmium Tetroxide

Expensive, not fast acting, used for electron microscopy. Volume: 5-10x the volume of specimen. Used to fixed myelin and peripheral nerves and for processing neurological tissues

Secondary Fixation

Placing an already fixed tissue to a second fixative to improve demonstration of substance, ensure complete hardening and for special staining.

Washing out

Removal of excess fixative, can use tap water, alcoholic iodine or 50-70% alcohol