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Questions and Answers
Which fixative is known for preventing the formation of formalin pigment?
Which fixative is known for preventing the formation of formalin pigment?
Why must tissue samples fixed in Zenker’s fluid be washed in running water after fixation?
Why must tissue samples fixed in Zenker’s fluid be washed in running water after fixation?
What is a major advantage of using Bouin’s Fluid for fixation?
What is a major advantage of using Bouin’s Fluid for fixation?
What volume ratio should be maintained between fixative and specimen for proper fixation?
What volume ratio should be maintained between fixative and specimen for proper fixation?
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When using 10% Formalin, how long can tissues be preserved safely?
When using 10% Formalin, how long can tissues be preserved safely?
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For which type of microscopy are glutaraldehyde and osmium tetroxide commonly used?
For which type of microscopy are glutaraldehyde and osmium tetroxide commonly used?
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What is the function of Lugol’s iodine in tissue processing?
What is the function of Lugol’s iodine in tissue processing?
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What procedure should be taken to ensure proper fixation of large specimens?
What procedure should be taken to ensure proper fixation of large specimens?
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What is the primary purpose of dehydration in tissue processing?
What is the primary purpose of dehydration in tissue processing?
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Which of the following methods is primarily used for clearing in tissue processing?
Which of the following methods is primarily used for clearing in tissue processing?
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What effect does insufficient dehydration have on the tissue during processing?
What effect does insufficient dehydration have on the tissue during processing?
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What is the main purpose of embedding tissue in paraffin wax?
What is the main purpose of embedding tissue in paraffin wax?
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When using a microtome, what is the likely remedy for obtaining sections with thin and thick horizontal lines, known as 'chatters'?
When using a microtome, what is the likely remedy for obtaining sections with thin and thick horizontal lines, known as 'chatters'?
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Which type of tissue processing fault would likely occur if air bubbles are present in the tissue or wax?
Which type of tissue processing fault would likely occur if air bubbles are present in the tissue or wax?
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Which statement best describes the role of histochemical stains in tissue processing?
Which statement best describes the role of histochemical stains in tissue processing?
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What could cause a tissue block to be too soft during trimming?
What could cause a tissue block to be too soft during trimming?
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What is the primary principle of fixation in histopathological examination?
What is the primary principle of fixation in histopathological examination?
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Which fixative is most commonly used in histopathological examination?
Which fixative is most commonly used in histopathological examination?
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What is a potential risk of using sit long-standing formalin for fixation?
What is a potential risk of using sit long-standing formalin for fixation?
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Why is absolute alcohol not commonly used as a primary fixative?
Why is absolute alcohol not commonly used as a primary fixative?
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What is the maximum time tissue should be kept in picric acid to avoid issues?
What is the maximum time tissue should be kept in picric acid to avoid issues?
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What does the use of osmium tetraoxide in fixation primarily benefit?
What does the use of osmium tetraoxide in fixation primarily benefit?
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How should formal saline be handled during tissue fixation?
How should formal saline be handled during tissue fixation?
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What is the recommended volume ratio of fixative to tissue for effective fixation?
What is the recommended volume ratio of fixative to tissue for effective fixation?
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Study Notes
Tissue Processing
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Aim: To make tissue rigid and thin enough to be cut into sections for microscopic examination
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Steps:
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Dehydration: Removing free or unbound water molecules from tissue using alcohol.
- Required because paraffin (the embedding medium) is not miscible with water.
- Graded alcohol is used (50%, 70%, 90%, and 100%).
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Clearing: Removing the dehydrating agent (alcohol) to make the tissue transparent for microscopic examination.
- Allows for infiltration of paraffin wax.
- Xylene is commonly used as a clearing agent.
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Impregnation/ Infiltration: Replacing the clearing agent with molten wax through diffusion.
- Parrafin wax is the most commonly used embedding medium.
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Embedding
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Purpose: To support the tissue, prevent distortion during cutting, and preserve the tissue for archival use
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Process:
- The processed tissue is placed in a mold and paraffin wax is poured around it.
Microtome
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Purpose: To cut the tissue block into thin sections (4-5 microns) for microscopic examination.
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Troubleshooting for Poor Sections:
- Chattering (thin and thick horizontal lines): Caused by a loose knife, block, or a blunt knife. Remedy: Tighten the knife and/or block, sharpen the knife.
- Holes in the section: Caused by air bubbles in the tissue or wax, a hard material in the tissue, or a soft piece of tissue in the block. Remedy: Re-embed, remove hard material if possible, reprocess the specimen.
- Tissue shrunken away from wax: Caused by insufficient dehydration. Remedy: Reprocess the specimen.
- Tissue too soft when block is trimmed: Caused by insufficient fixation. Remedy: Reprocess the specimen.
Histochemical Stain
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Purpose: To color the colorless tissue sections to enable microscopic examination.
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Principle: Different tissues have different affinities for different stains, allowing them to be distinguished visually.
Fixation
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Principle: To prevent autolysis and putrefaction of tissues by denaturing and coagulating proteins.
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Aim: To preserve tissue structure as close to its living state as possible.
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Common Fixative: 10% neutral buffered formalin
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Important Considerations:
- Mineralized samples (bone, teeth) may require decalcification before processing.
- The volume of the fixative should be at least 20 times the volume of the tissue.
- Tissues are typically fixed for 6 to 24 hours.
Common Simple Fixatives
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Formaldehyde: Most commonly used fixative.
- Cheap, penetrates rapidly, and does not over-harden tissues.
- Buffered formalin is recommended to prevent the formation of formalin pigment.
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Absolute Alcohol: Coagulates proteins, but can cause shrinkage and distortion due to rapid dehydration.
- Used for cytological specimen fixation.
- Osmium Tetraoxide: Provides excellent preservation of cellular details, used for electron microscopy.
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Picric Acid: Precipitates proteins, forming picrates.
- Explosive when dry; must be kept under water.
- Requires thorough washing to remove color.
- Cannot be kept in for more than 24 hours.
Common Compound Fixatives
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Formal Saline: Most widely used fixative, tissues can be left in this for long periods.
- May cause formalin pigment formation with prolonged storage.
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Zenker's Fluid: Contains mercuric chloride, potassium dichromate, sodium sulfate, and acetic acid.
- Provides even penetration and rapid fixation.
- Requires thorough washing in running water to remove excess dichromate and removal of mercury pigment with Lugol’s iodine.
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Zenker’s Formal (Helly’s Fluid): Formalin replaces acetic acid in stock Zenker’s fluid.
- Excellent fixative for microanatomical structures, especially bone marrow, spleen, and kidney.
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Bouin’s Fluid: Contains picric acid, acetic acid, and 40% formaldehyde.
- Provides rapid and even penetration with no shrinkage.
- Excellent for trichrome staining.
- Routinely used for preserving testicular biopsies.
Most Commonly Used Fixatives in the Laboratory
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10% Formalin: 10 ml of 40% formaldehyde mixed with 90 ml of distilled water.
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Formal Saline: 100 ml of 40% formaldehyde mixed with 900 ml of distilled water and 9 grams of sodium chloride.
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10% Buffered Formalin: 10 ml of 40% formaldehyde mixed with 90 ml of distilled water, 0.4 grams of sodium dihydrogen phosphate, and 0.65 grams of disodium hydrogen phosphate.
- Prevents the formation of formalin pigment.
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Bouin’s Solution: 750 ml of saturated picric acid (1.2 g/100 ml), 250 ml of 40% formaldehyde, and 50 ml of acetic acid.
Points to Remember
- 10% buffered formalin is the most common fixative.
- Tissues can be kept in 10% buffered formalin for extended periods.
- The volume of the fixative should be at least ten times the volume of the specimen.
- Special fixatives are used for preserving specific tissues.
- Formalin vapors are irritating; always wear a mask, eye protection, and gloves.
- Tissues should be well-fixed before dehydration.
- Fixative penetration takes time; larger specimens should be cut to ensure fixation of the central part.
- Mercury pigment must be removed with Lugol’s iodine.
- Bouin’s fluid should not be used for more than 24 hours without changing the alcohol.
- Glutaraldehyde and osmium tetraoxide are used for electron microscopy fixation.
Histopathological Examination
- Routine Hematoxylin and Eosin Stain: Basic stain used to visualize tissue morphology.
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Ancillary Techniques: Used to further investigate tissue characteristics.
- Special Stains: Specific stains that highlight particular tissue elements.
- Immunohistochemistry: Uses antibodies to detect specific proteins in tissues.
- Immunofluorescence: Uses fluorescently labeled antibodies to detect specific proteins in tissues.
- Electron Microscopy: Uses electron beams to visualize ultrastructural details of cells and tissues.
- Molecular Studies: Analyze genetic material (DNA and RNA) in tissues.
- Cytogenetic Studies: Study chromosomes and their abnormalities.
- Flow Cytometry: Measures the properties of individual cells in suspension.
- Polymerase Chain Reaction (PCR): Amplifies DNA or RNA for analysis.
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Description
This quiz explores the essential techniques for tissue processing and embedding, crucial for microscopic examination. It covers steps like dehydration, clearing, and infiltration using paraffin wax, as well as the role of the microtome in sectioning. Test your knowledge of these vital laboratory procedures.
