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Questions and Answers

What effect does rising temperature have on the diffusion of molecules?

  • It causes diffusion to stop completely.
  • It increases the diffusion rate. (correct)
  • It decreases the diffusion rate.
  • It has no impact on diffusion.

Which concentration of formalin tends to increase hardening and shrinking of tissues?

  • Exactly 10%
  • Above 10% (correct)
  • Less than 5%
  • From 5% to 10%

What is the effect of a hypertonic fixative on fixed tissue?

  • It causes the tissue to shrink. (correct)
  • It causes the tissue to swell.
  • It makes the tissue more soluble.
  • It leads to no significant changes.

Which additive is said to improve the morphology of fixed tissue?

<p>Calcium chloride (A)</p> Signup and view all the answers

What type of pigment is produced from fixation in mercuric chloride?

<p>Mercuric pigments (C)</p> Signup and view all the answers

What is the primary advantage of using Gluteraldehyde as a cross-linking fixative?

<p>It preserves subcellular structures for electron microscopy. (A)</p> Signup and view all the answers

Which fixative is specifically known for lipid fixation and contrast enhancement of cell membranes?

<p>Osmium Tetroxide (A)</p> Signup and view all the answers

What is a significant disadvantage of the fixation process?

<p>Potential swelling or shrinkage of specimens. (D)</p> Signup and view all the answers

How does pH affect the quality of fixation?

<p>An acid pH can cause insoluble pigment formation. (C)</p> Signup and view all the answers

Which of the following fixatives is often combined with formaldehyde to improve tissue density for light microscopy?

<p>Mercuric Chloride (C)</p> Signup and view all the answers

What is the relationship between the duration of fixation and the depth of penetration by the fixative?

<p>Directly proportional; increases with the square root of duration. (D)</p> Signup and view all the answers

Which of the following options includes a type of compound fixative?

<p>Zenker's solution (C)</p> Signup and view all the answers

What effect does alcohol added to formaldehyde produce?

<p>Produces alcoholic formalin that preserves glycogen. (B)</p> Signup and view all the answers

Flashcards

Glutaraldehyde Fixation

A cross-linking fixative ideal for ultrastructural studies, preserving fine cellular details quickly.

Osmium Tetroxide Fixation

A fixative that binds to lipids, preserving and contrasting cell membranes, often used after aldehyde fixation for better ultrastructure preservation.

Mercuric Chloride Fixation

A fixative excellent for preserving protein structures and maintaining tissue morphology, enhancing tissue staining for light microscopy.

Compound Fixatives

Formaldehyde-based fixatives that may include additional agents for specific effects, like preserving glycogen (e.g. Carnoy, Zenker, Susa).

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Fixation Disadvantages

Fixation can cause molecular loss, tissue swelling/shrinkage, staining variations, and loss of antigen recognition.

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Fixation Quality Factors

Factors like pH, duration, and specimen size affect fixation quality. Using neutral buffered formalin (NBF) is important to avoid artifacts like pigment formation.

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Light Microscopy

A microscopy technique using visible light to view samples, often used in pathology.

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Electron Microscopy

Microscopy technique using a beam of electrons to view samples, often used in pathology.

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Diffusion of molecules

Movement of molecules increases with rising temperature.

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Fixative Concentration

Appropriate fixative concentration is crucial; exceeding 10% formalin can over-harden tissues, while below 70% ethanol may not effectively remove water.

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Osmolality and tissue fixation

Hypertonic fixatives cause tissue shrinkage, while hypotonic fixatives cause swelling. Ideal osmolality is slightly hypertonic (400-450 mosm).

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Fixative Additives

Electrolytes (like calcium chloride) and non-electrolytes (like sucrose) can improve tissue morphology during fixation. However, electrolytes may also cause protein denaturation.

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Fixation Artifacts

Artifacts like formalin pigment (removed by picric acid), mercuric pigment (removed by alcohol), or chrome oxide pigment (removed by acid alcohol) occur during fixation and can affect tissue morphology.

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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

  • pH:
    • Formalin in acidic pH causes brown-black insoluble crystalline pigment formation. Use neutral buffer formalin (NBF) to avoid this.
  • Duration and size of specimens:
    • Depth reached by fixatives is directly proportional to the square root of duration ( d=k √ t)
  • Temperature:
    • Molecule diffusion increases with rising temperature.
  • 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.
  • 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).
  • Additives:
    • Electrolytes and non-electrolytes improve fixed tissue morphology. (examples: calcium chloride, potassium thiocyanate, ammonium sulfate, sucrose, dextran)

Fixative Artifacts

  • Formalin Pigments:
    • Produced from fixation of non-buffered formalin
    • Removed by saturated alcoholic picric acid
  • Mercuric Pigments:
    • Produced from fixation in mercuric chloride
    • Removed by alcohol
  • Chrome oxide pigments:
    • Produced from fixation in chrome acid
    • Removed by 1% acid alcohol

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