Histology Lecture 1: Micro Techniques

Study Notes

Histology - Lecture 1

Micro technique: Techniques used to prepare tissue samples for microscopic examination.
- Paraffin technique: A common technique for preparing tissue for histological examination.
  - Advantages: Easy to use and provides good tissue preservation.
  - Issues: Paraffin can melt at high temperatures, potentially damaging tissue.
- Frozen technique: Used for rapid tissue preparation.
- Colloidin technique: A technique that employs colloidin, a solution that hardens tissue for sectioning.

Paraffin Technique: Steps

Tissue sample: The first step is to obtain a tissue sample. It is important to ensure that the sample is handled carefully and quickly to prevent tissue deterioration.
Fixation:
- The most common fixative used in histology is 10% formaldehyde.
- Purpose:
  - It preserves the fine structure of tissues, preventing deterioration.
  - It inhibits the activity of lysosomal enzymes, which can cause autolysis (self-destruction of cells).
  - It kills bacteria, preventing putrefaction (decomposition).
  - It hardens tissues, making them easier to cut.
Dehydration: Removal of water from the tissue sample using a series of graded alcohols to prevent tissue damage.
Clearing: The tissue sample is then transferred to a paraffin solvent. Commonly, xylene or other benzene derivatives are used.
Impregnation: The tissue sample is placed in melted paraffin, which penetrates the tissue, replacing the clearing agent.
Embedding: The tissue sample is placed in a mold filled with melted paraffin, which solidifies into a block.
Sectioning: The paraffin block is cut into thin sections using a microtome (5-8 µm thick).
Mounting: The tissue sections are then mounted on glass slides.

Staining Techniques

1) Basic & Acidic Stains:
- Hematoxylin & Eosin (H&E): A common stain used in histology.
- Hematoxylin is a basic dye, while Eosin is an acidic dye. This means Hematoxylin stains basophilic structures, while Eosin stains acidophilic structures.
  - Basophilic: Affinitive to basic stains. These structures include:
    - Nucleus (due to DNA & RNA)
    - Ribosomes
  - Acidophilic: Affinitive to acidic stains. These structures include:
    - Cytoplasm
    - Muscle
- Common Basic Stains: Hematoxylin, Toluidine Blue
- Common Acidic Stains: Eosin, Orange G
2) Neutral Stains:
- These stains are made of a combination of a basic dye and an acidic dye.
- They can stain different tissue structures depending on their pH.
- Example: Leishman's Stain
3) Vital Stains:
- Staining methods used for living cells inside the living body.
- Example: Trypan blue. This dye is taken up by phagocytic cells, which are responsible for engulfing foreign bodies.
4) Supravital Stains:
- Staining of living cells outside the body.
- Example: Janus Green B Stain, which targets mitochondria.
5) Metachromatic Stain:
- A certain cell component stains with a color different from the original dye.
- Example: Mast cells stained with Toluidine Blue appear red due to their high protein content. This phenomenon is called a metachromatic reaction.
6) Histochemical Stain:
- Used to identify specific chemical structures within cells.
- Examples:
  - PAS Staining: Used to stain glycogen (carbohydrates) in liver cells.
  - Sudan III: Used for staining fats.
- Note: Carbohydrates and Lipids are often dissolved during the paraffin technique because of the heating process (50°/60°C).
7) Immunohistochemistry:
- Used to identify and localize specific proteins using antibodies.
8) Special Stains:
- These are specialized stains used to differentiate between specific cell types or structures.
- Example: Silver stain, which stains the Golgi apparatus brown and in color.

Microscopes

Light Microscope:
- Uses a beam of visible light to illuminate the sample.
- Produces colored images.
- Cannot resolve very small structures within cells.
Electron Microscope:
- Uses a beam of electrons to illuminate the sample.
- Produces black and white images.
- Provides high resolution, allowing visualization of fine details within cells.
- Transmission Electron Microscopy (TEM): Produces two-dimensional (2D) images.
- Scanning Electron Microscopy (SEM): Produces three-dimensional (3D) images.