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Questions and Answers
What effect does rising temperature have on the diffusion of molecules?
What effect does rising temperature have on the diffusion of molecules?
Which concentration of formalin tends to increase hardening and shrinking of tissues?
Which concentration of formalin tends to increase hardening and shrinking of tissues?
What is the effect of a hypertonic fixative on fixed tissue?
What is the effect of a hypertonic fixative on fixed tissue?
Which additive is said to improve the morphology of fixed tissue?
Which additive is said to improve the morphology of fixed tissue?
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What type of pigment is produced from fixation in mercuric chloride?
What type of pigment is produced from fixation in mercuric chloride?
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What is the primary advantage of using Gluteraldehyde as a cross-linking fixative?
What is the primary advantage of using Gluteraldehyde as a cross-linking fixative?
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Which fixative is specifically known for lipid fixation and contrast enhancement of cell membranes?
Which fixative is specifically known for lipid fixation and contrast enhancement of cell membranes?
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What is a significant disadvantage of the fixation process?
What is a significant disadvantage of the fixation process?
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How does pH affect the quality of fixation?
How does pH affect the quality of fixation?
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Which of the following fixatives is often combined with formaldehyde to improve tissue density for light microscopy?
Which of the following fixatives is often combined with formaldehyde to improve tissue density for light microscopy?
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What is the relationship between the duration of fixation and the depth of penetration by the fixative?
What is the relationship between the duration of fixation and the depth of penetration by the fixative?
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Which of the following options includes a type of compound fixative?
Which of the following options includes a type of compound fixative?
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What effect does alcohol added to formaldehyde produce?
What effect does alcohol added to formaldehyde produce?
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Study Notes
Histopathology Techniques - Fixation
- Glutaraldehyde is a cross-linking fixative
- Ideal for ultrastructural studies due to detailed preservation
- Rapid fixation, cross-linking achieved quickly, ensuring fine cellular detail
- High fixation strength preserves subcellular structures (organelles and membranes), making it optimal for electron microscopy
- Osmium Tetroxide fixatives have properties related to lipid fixation and secondary fixation
- Binds to unsaturated lipids, preserving and contrasting cell membranes
- Often used after aldehyde-based fixation for better ultrastructure preservation
- Staining Properties:
- Give strong electron density and dark-staining cellular structures for visibility.
- Mercuric Chloride
- Excellent for preserving protein structures and maintaining tissue morphology
- Adds density, enhancing tissue staining for light microscopy
- Often combined with other fixatives (like formalin) in Zenker's and Helly's solutions
- Compound fixative:
- Pathologists use formaldehyde-based fixatives to preserve histomorphometric patterns
- Other agents may be added to formaldehyde to produce specific effects not possible with formaldehyde alone
- Ethanol added to formaldehyde produces alcoholic formalin to preserve molecules like glycogen
- Examples include Carnoy fixative, Susa, and Zenker fixatives
Disadvantages of Fixative
- Molecular loss
- Swelling or shrinkage
- Variation in the quality of biochemical and immunohistochenmical stain
- Loss of antigen immunorecognition
Factors Affecting Fixation Quality
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pH:
- Formalin in acidic pH causes brown-black insoluble crystalline pigment formation. Use neutral buffer formalin (NBF) to avoid this.
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Duration and size of specimens:
- Depth reached by fixatives is directly proportional to the square root of duration ( d=k √ t)
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Temperature:
- Molecule diffusion increases with rising temperature.
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Concentration of fixative:
- Effectiveness and solubility determine appropriate concentration.
- Formalin above 10% tends to cause hardening and shrinking.
- Ethanol below 70% does not remove water from tissue efficiently.
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Osmolality and ionic composition:
- Hypertonic fixative leads to shrinking, hypotonic fixative to tissue swallowing.
- Optimal morphological result is with slightly hypertonic solution (400-450 mosm).
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Additives:
- Electrolytes and non-electrolytes improve fixed tissue morphology. (examples: calcium chloride, potassium thiocyanate, ammonium sulfate, sucrose, dextran)
Fixative Artifacts
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Formalin Pigments:
- Produced from fixation of non-buffered formalin
- Removed by saturated alcoholic picric acid
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Mercuric Pigments:
- Produced from fixation in mercuric chloride
- Removed by alcohol
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Chrome oxide pigments:
- Produced from fixation in chrome acid
- Removed by 1% acid alcohol
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