Histology and Micro-techniques

Histology

• The study of the structure of cells and tissues using a microscope
• It is important to correlate structure with function and understand the pathology of diseases

Micro-techniques

• Micro-techniques encompass the methods employed in preparing and examining tissue samples for histological analysis.
• There are two main techniques for preparing thin sections: Paraffin technique and Freezing technique

Paraffin technique

• Routine method for histological preparation
• Involves several steps to preserve, dehydrate, clear, and embed the tissue in paraffin wax for sectioning
• Steps include:
  • Obtaining a sample
  • Fixation: using formalin to prevent putrefaction and autolysis
  • Dehydration: using ascending grades of alcohol (70%-90%-100%) to avoid tissue shrinkage
  • Clearing: using xylene to replace alcohol, render the tissue transparent, and prepare it for paraffin infiltration
  • Impregnation: infiltrating the tissue with paraffin wax in an oven
  • Embedding in paraffin: placing the impregnated tissue in hard paraffin for block formation
  • Sectioning: using a rotary microtome to create thin sections (5um thick)
  • Picking the section on a slide

Freezing technique

• Quick method used for rapid diagnosis in operating theatres
• Preserves lipids and enzymes for histochemistry
• Employs cryocut for freezing the specimen

Staining

• Used to enhance the visibility of subtle structures in tissue sections

Ordinary histological stain

• Hematoxylin and Eosin (H&E)
• Hematoxylin stains basic structures (nucleus) blue, and Eosin stains acidic structures (cytoplasm) pink

Special stains

• Used to highlight specific components or structures within the tissue

• Carbohydrates:

  • Periodic acid Schiff’s technique (PAS): stains carbohydrates magenta
  • Best's carmine: stains glycogen red

• Lipids:

  • Frozen sections are used:
    • Sudan III: stains lipids orange
    • Sudan black: stains lipids black
    • Osmic acid: stains lipids black

• Vital stain:

  • Involves staining living cells in vivo by injecting dyes like Trypan blue, e.g., for phagocytic cells

• Supravital stain:

  • Involves staining living cells in vitro outside the body, e.g., mitochondria by Janus green B

• Metachromatic stain:

  • Involves staining with a color different from the color of the dye, e.g., granules of mast cells are stained red when stained with toluidine blue

Microscopy

• The microscope is an instrument that magnifies the image and reveals fine details of an object.
• Types of microscopes:

Light Microscopy

• Magnification: up to 1000 times
• Types: Student, Compound, Stereo

Electron Microscopy

• Magnification: up to 100,000 times
• Types:
  • Transmission Electron Microscopy (TEM):
    • Utilizes an electron beam for illumination
    • Creates black-and-white images
    • Dark components are electron-dense, and white components are electron-lucent
    • Requires extremely thin sections (0.08 um)
  • Scanning Electron Microscopy (SEM):
    • Provides three-dimensional images of the surface of the specimen

Other types of microscopes

• Phase Contrast Microscope: used to study living or fixed unstained cells
• Fluorescence Microscope: used to detect fluorescent substances

Case Scenario

• A 45-year-old female patient presents with a swelling in her neck
• Clinical examination reveals a firm, non-tender, but fully mobile mass
• Ultrasound suggests the mass may be malignant
• A biopsy is needed for a proper diagnosis

Summary

• Histology is the study of the structure of cells and tissues, correlating structure with function and understanding disease pathology.
• Micro-techniques for tissue processing include paraffin and freezing techniques.
• Common histological stains include H&E, with special stains highlighting specific structures.
• Microscopes, including light and electron microscopes, are vital tools for visualizing cellular and tissue details.

Explore the fascinating world of histology, the study of cell and tissue structures using microscopy. This quiz covers key micro-techniques for preparing tissue samples, focusing on the paraffin method. Test your knowledge on the steps involved in histological preparation and the importance of structure-function correlation.