Chapter 6: Types and Mechanisms of Fixation

What are the two main classifications of fixatives according to composition?

Simple Fixatives (one component) and Compound Fixatives (two or more components).

What are some examples of compound fixatives?

Aldehyde Fixatives and Metallic Fixatives.

What are the types of fixatives according to action?

Mercuric Chloride, Chromate, Lead Fixatives, Picrate Fixatives.

What is the purpose of microanatomical fixatives?

They permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationships.

What do cytological fixatives preserve?

They preserve specific parts and particular microscopic elements of the cell.

What is the primary component of nuclear fixatives?

Glacial acetic acid.

What is the pH requirement for cytoplasmic fixatives?

pH > 4.6.

What is the primary goal of secondary fixation?

To preserve the morphologic and chemical integrity of the cell in a life-like manner.

What is the ideal time to perform fixation after interruption of blood supply?

20 - 30 minutes.

What is the recommended fixative to tissue ratio?

20:1.

What is additive fixation?

A process where the chemical constituents of the fixative become part of the tissue, forming cross-links or molecular complexes.

What is the purpose of post-chromatization in fixation?

It aids in cytologic preservation of tissues by placing fixed tissue in a potassium dichromate solution.

What is non-additive fixation?

A process where tissue composition is altered and stabilized through the removal of bound water.

What is the aim of fixation for enzyme histochemistry?

To preserve maximum enzyme activity at its original localization while maintaining structural integrity.

What is the process of washing out in fixation for enzyme histochemistry?

Removing excess fixative from the tissue after fixation to improve staining and remove artifacts.

What is the role of 50-70% alcohol in fixation for immunofluorescence?

It is used to wash out excess amounts of picric acid.

What is the effect of glacial acetic acid in cytoplasmic fixatives?

It destroys mitochondria and Golgi bodies.

What is the significance of fixation artifacts in pathology?

They are commonly used for the demonstration of various antibodies.

What are some examples of fixatives used in fixation for immunofluorescence?

Formalin, Mercury, Osmium tetroxide.

What is the purpose of fixation in histotechnology?

To preserve tissues from decay and prevent autolysis and putrefaction.

What is the effect of alcoholic fixatives in non-additive fixation?

They stabilize tissue by removing bound water.

What is the primary component of formalin used in fixation?

Formaldehyde.

What is the role of secondary fixatives in histotechnology?

They act as mordants and ensure complete hardening and preservation of tissues.

What is the primary use of immunohistochemistry in pathology?

To demonstrate various antibodies.

What type of sections are commonly used for immunohistochemistry?

Formalin-fixed and paraffin embedded sections.

What techniques are required for antigen retrieval in immunohistochemistry?

Antigen-retrieval techniques.

What is the preferred type of sections for immunohistochemistry?

Frozen sections.

What is crush artifact and where is it commonly found?

Crush artifact is found in surgical specimens, such as liver biopsies, and may be due to partial coagulation of proteins or incomplete wax impregnation.

What is the purpose of lipid fixation in histology?

To fix target cells to the slide and preserve cellular architecture.

What are organic solvents used for in lipid fixation?

They remove lipids and dehydrate cells while precipitating proteins.

What is the role of cross-linking reagents in lipid fixation?

They form intermolecular bridges to preserve cell structure better than organic solvents but may reduce antigenicity.

What is the function of a cryostat in lipid fixation?

It is used for demonstrating lipids in tissues.

Which fixatives preserve lipids in cryostat sections?

Fixatives containing mercuric chloride and potassium dichromate.

What is the fixation method for carbohydrates using acetone?

Fix cells in -20°C acetone for 5-10 minutes without a permeabilization step.

What is the fixation method for carbohydrates using methanol?

Fix cells in -20°C methanol for 5-10 minutes, requiring a permeabilization step.

What are the recommended fixatives for glycogen fixation?

Alcoholic fixatives, particularly alcoholic formaldehyde.

What is the common fixative used for amino acid histochemistry?

Neutral buffered formal saline (formaldehyde vapor).

What is the recommended fixation procedure for electron microscopy?

Perform fixation at 4°C, permeabilize with cooled acetone, and fix in cooled methanol.

What is the composition of the routine fixation mixture for electron microscopy?

A 1:1 mixture of methanol and acetone.

What is the purpose of Karnovsky's paraformaldehyde-glutaraldehyde mixture?

It is used for electron histochemistry and electron immunocytochemistry.

What is the application of formalin fixation in histopathology?

It is used for routine histopathology and preservation of lipids.

What is the formula for preparing paraformaldehyde for fixation?

40% formaldehyde: 100 mL, distilled water: 900 mL, sodium dihydrogen phosphate: 4 gm, disodium hydrogen phosphate: 6.5 gm, pH: 6.8.

What are the advantages of methanol fixation?

It penetrates and fixes tissues evenly, preserves enzymes and nucleoproteins, and demonstrates fats and mucin.

What are the disadvantages of methanol fixation?

It may reduce the metachromatic reaction of amyloid and acid dye stains less brightly compared to mercuric chloride fixation.

What are the two classifications of fixatives?

Physical fixatives and chemical fixatives.

What is the most commonly used physical fixative?

10% neutral-buffered formalin.

What is the primary application of chemical fixatives?

For post-mortem and research specimens.

What are the two types of chemical fixatives?

Simple and Compound.

What are the two categories of compound fixatives?

Microanatomical and Cytological.

What is the formula for preparing 10% neutral-buffered formalin?

6.5 gm Sodium Dihydrogen phosphate (anhydrous), 4 gm Sodium Dihydrogen Phosphate * H2O, 900 mL Distilled water, pH 7.4, and 100 mL 40% formaldehyde.

What are the two main components of cellular structures that can be fixed?

Nuclear and Cytoplasmic.

What is the best fixative for tissue containing iron pigments?

Formaldehyde/Formalin.

What are the advantages of using Formaldehyde/Formalin as a fixative?

It prevents precipitation of acid formalin pigments, requires no post-treatment after fixation, and can go directly into 80% alcohol for processing.

What are the disadvantages of Formaldehyde/Formalin?

It takes longer to prepare, may cause gradual loss in basophilic staining of cells, and is inert towards lipids.

What is the fixation time for Formol-Corrosive (Formol-Sublimate)?

3 to 24 hours.

What are the advantages of using Formol-Corrosive (Formol-Sublimate)?

It is cheap, easy to prepare, and readily available.

What is a disadvantage of Formol-Corrosive (Formol-Sublimate)?

It may produce considerable shrinkage of tissues.

What is the fixation time for 10% Formal-Saline?

12 to 24 hours.

What are the advantages of 10% Formal-Saline?

It penetrates small tissues, produces minimum shrinkage and hardening, brightens cytoplasmic and metachromatic stains, and requires no washing out.

What is a disadvantage of 10% Formal-Saline?

It has long-term storage issues and may not penetrate tissues well.

What are the characteristics of Karnovsky's Fixative?

It forms mercuric chloride deposits, does not allow frozen tissue sections, and inhibits determination of tissue decalcification extent.

What is the application of Glutaraldehyde in histochemistry?

It is used for light microscopy in resin embedding and sectioning, and for electron microscopy.

What is the formula for preparing a 2.5% solution of Glutaraldehyde?

For small tissue fragments and needle biopsies, fixed in 2-4 hours at room temperature.

What are the advantages of Mercuric Chloride as a fixative?

It preserves cellular structures, produces less tissue shrinkage, and preserves plasma proteins.

What is a disadvantage of Mercuric Chloride?

It may produce black granular deposits on tissues.

What are the characteristics of Zenker's Fluid?

It is more expensive, less stable, penetrates slower, and can brittle the tissue.

What is the recommended fixation time for Zenker's Fluid?

12 to 24 hours.

What is the application of Zinc Formalin (unbuffered)?

It is an alternative to mercuric chloride formulation.

What are the advantages of using Paraformaldehyde?

It produces rapid and even fixation, permits brilliant staining, is compatible with most stains, and is stable for many years.

What is a disadvantage of Paraformaldehyde?

It has poor penetration and can cause lysis of RBC.

What is the application of Zenker's-Formol (Helly's) Solution?

After using, tissue should be transferred directly to a high-grade alcohol to avoid undue swelling.

What is the fixation time for Lillie's B-5 Fixative?

4 to 24 hours.

What are the applications of Lillie's B-5 Fixative?

It is used for bone marrow, extra medullary hematopoiesis, intercalated discs of cardiac muscle, spleen, liver, and pituitary gland.

What are the advantages of Lillie's B-5 Fixative?

Rapid fixation can be achieved in 1 ½ - 2 hours, and it produces excellent nuclear detail.

What is a disadvantage of Lillie's B-5 Fixative?

Overfixation can harden the tissue and make cutting difficult.

What happens if tissues are allowed to stay in chromate fixative for more than 24 hours?

Brown pigments are produced due to RBC lysis.

What is the primary purpose of chromic acid in tissue preservation?

To preserve chromatin.

How can chromic acid be removed from tissue samples?

By immersing the tissue in saturated alcoholic picric acid or sodium hydroxide.

What are the key features of Heidenhain's Susa solution?

Used in 1-2% solution, preserves carbohydrates, and is a strong oxidizing agent.

What is the fixation time for potassium dichromate?

3 to 12 hours.

What does potassium dichromate preserve in tissues?

Mitochondria and lipids.

What is the recommended application of potassium dichromate?

For tumor biopsies, especially of the skin, and it is an excellent cytologic fixative.

What is the formula for Regnaud's (Muller's) fixative?

Mercuric Chloride: 45 gm, Sodium chloride: 5 gm, Trichloroacetic acid: 20 gm, Glacial acetic acid: 40 ml, 40% Acid formaldehyde: 200 ml, 40% Distilled water: 800 ml.

What are the advantages of using Regnaud's (Muller's) fixative?

Recommended for chromatin, mitochondria, Golgi apparatus, and RBC; penetrates and fixes tissues rapidly and evenly; produces minimum shrinkage and hardening.

What are the disadvantages of Regnaud's (Muller's) fixative?

Prolonged fixation blackens tissue and produces precipitates of suboxide.

What is the composition of Brasil's Alcoholic Picrofomol?

Potassium dichromate (3%) + formaldehyde (40%).

What is Orth's fluid used for?

For studying Rickettsia and early degenerative diseases.

What is the fixation time for Orth's fluid?

36 to 72 hours.

What is the primary advantage of lead fixative?

It fixes connective tissue mucin.

What is a disadvantage of lead fixative?

It forms insoluble lead carbonate, which must be removed by filtration or by lowering pH with acetic acid.

What is Alcian's Blue used for?

For staining acid mucopolysaccharides.

What is a key characteristic of picrate fixative?

It freezes and solidifies at 16°C and precipitates DNA, making it valuable for preserving nuclei.

What is the disadvantage of picrate fixative when combined with GAA?

It destroys the lipid-fixing property of K2Cr2O7 and damages mitochondria and Golgi elements.

What is the importance of alcohol fixative in tissue preparation?

It precipitates proteins and is essential for preserving glycogen.

What is the recommended use of Bouin's solution?

For fixing thin layer preparations such as blood films or cell cultures.

What is the fixation time for methyl alcohol (100%)?

4 to 18 hours.

What are the advantages of using methyl alcohol for fixation?

Excellent for preserving soft and delicate tissues, such as gastrointestinal tract biopsies and endocrine gland tissues.

What is a disadvantage of methyl alcohol in tissue fixation?

It penetrates large tissues poorly and can cause hemolysis.

What is the effect of prolonged fixation with methyl alcohol?

Tissue hardens and becomes difficult to cut if left for more than 48 hours.

What is the primary use of Isopropyl Alcohol (95%)?

Used for fixing touch preparation.