Tissue Preparation Techniques Quiz

Tissue Preparation Techniques

• Paraffin method: A popular and quick technique for preparing tissue sections.
  • Uses a series of solutions with increasing concentrations of paraffin to dehydrate and embed the tissue
  • Paraffin allows for thin sections and easy staining
  • Disadvantages: can damage tissues, dissolve fats, not ideal for histochemistry or large specimens.
• Celloidin method: Preferred for larger specimens with complex structures.
  • Embeds tissues in a celloidin block for sectioning.
  • No heat used, preserving tissue integrity.
  • Disadvantages: Time-consuming, no serial sections, and difficult to stain.
• Freezing technique: For quick analysis of fresh specimens.
  • Tissues are frozen, hardened, and cut using a cryostat.
  • Useful for surgical procedures and enzyme studies where heat inactivation is undesirable.
  • Disadvantages: Thick sections, no serial sections, and potential fragmentation of the specimen.

Electron Microscopy Specimen Preparation

• Tissues are fixed in glutaraldehyde followed by osmium tetroxide for preservation.
• Cleared in propylene oxide to prepare for epoxy resin embedding.
• Embedding in epoxy resin (like araldite or Epon) creates hard blocks for ultra-thin sectioning.
• Ultra-thin sections are cut using a diamond knife on an ultramicrotome (0.005-0.1 µm thickness).

Histochemistry & Cytochemistry

• Methods to identify and localize specific substances in tissue sections using light or electron microscopy.

Demonstration of Nucleic Acids

• Feulgen reaction: Detects DNA in cell nuclei, producing a red color.
• Methyl green Pyronin: Differentiates DNA (stains green) and RNA (stains red) in cells or tissue sections.

Demonstration of Enzymes

• Phosphatases:
  • Alkaline phosphatases: Active at alkaline pH.
  • Acid phosphatases: Found in lysosomes.
• Dehydrogenases: Such as succinate dehydrogenase (involved in the Krebs cycle), found in mitochondria.
• Peroxidase: Found in blood cells.

Demonstration of Glycogen

• Best's Carmine: Specific stain for glycogen, producing a red color.
• Periodic acid–Schiff (PAS) reaction: Produces a purple or magenta color, staining polysaccharides, oligosaccharides, and glycoproteins (PAS positive).

Demonstration of Lipids

• Sudan III: Stains lipids orange.
• Sudan black: Stains lipids black.
• Osmic acid: Stains lipids black.

Immunocytochemistry

• Identifies cellular or tissue constituents (antigens) using antigen-antibody interactions.

Microscopy

• Resolving Power: The ability of a microscope to distinguish between two closely spaced objects. The human eye's limit is 0.1 mm.

• Light microscopes: use visible light to illuminate the specimen.

  • Conventional light microscope: Uses a condenser, objective lens, and eyepiece lens to magnify the image.
  • Polarizing microscope: Uses polarized light to identify birefringent structures (e.g., muscle fibers, collagen fibers).
  • Phase contrast microscope: Uses phase plates that convert light speed differences into intensity differences, revealing subtle details.
  • Dark-field microscope: Uses a cardioid condenser to only allow scattered light from the specimen to reach the objective, producing a bright image against a dark background.
  • Fluorescence microscope: Uses UV light to excite fluorescent substances, producing their characteristic colors.

• Other microscopes: Use non-visible light.

  • X-ray microscope: Uses X-rays for high resolution imaging.
  • Laser microscopes:**
    • Confocal microscope: Uses a pinhole to focus on a specific plane of the specimen, producing a 3D image.
    • Multiphoton (Femto-second) microscope: Uses multiple photons to excite fluorescence deep within the specimen.

• Electron microscopes: provide much higher resolutions and magnification.

  • Transmission Electron Microscope (TEM): Transmits electrons through the thinly sliced specimen to create an image of the internal structure.
  • Scanning Electron Microscope (SEM): Scans a focused electron beam across the specimen and uses the emitted secondary electrons to create a 3D image of the surface structure.

Test your knowledge on various tissue preparation techniques including the paraffin method, celloidin method, and freezing technique. Understand their applications, advantages, and disadvantages in histology. This quiz is essential for students in biology or medical fields focused on tissue analysis.