Cytology · MU Pleven

Topic 3. Methods of cell study – preparation of permanent histological slide.

Walks through the steps for preparing a permanent histological slide, starting with fixation and its purposes. It covers chemical fixatives used in light and electron microscopy, freezing as a physical method, and heat fixation used in cytology. Tissue collection, rinsing, dehydration through graded ethanol, clearing, paraffin infiltration and embedding, sectioning, staining and mounting follow in order.

Inside: 3 sections · 2 labelled pictures · 12 flashcards · 2 min

Topic 3 of 59 in the MU Pleven syllabus for Cytology (Medicine, Year 1). The free AceMed note covers it in 3 sections, about 350 words (2 min of reading), with 12 flashcards.

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  1. TOPIC 3. Methods of cell study – preparation of permanent histological slide.
  2. Fixation
  3. Types of Fixation:

Fixation

Fixation is a crucial step that stabilizes and preserves the tissue structure by preventing enzymatic degradation. It involves various methods depending on the type of microscopy and the cellular components being examined.

Types of Fixation:

1. Chemical Fixation:

  • Involves the use of chemical agents (fixatives) to preserve tissue.
  • Formalin (37% formaldehyde) is a widely used fixative in light microscopy. It reacts with amine groups in proteins, preventing degradation.
  • Glutaraldehyde is commonly used in electron microscopy due to its protein cross-linking properties.
  • Osmium tetroxide is used for electron microscopy, especially for preserving and staining membrane lipids and proteins.
  • Heavy metal fixatives like lead citrate and uranyl compounds enhance contrast in transmission electron microscopy (TEM) by increasing electron density.

2. Physical Fixation:

  • Methods such as freezing preserve tissue by rapid cooling, commonly used for histochemical studies where enzyme activity needs to be preserved.

3. Heat Fixation:

  • Often used in cytology, where cells are fixed by briefly heating the slide to kill the cells and adhere them to the slide.

Tissue Collection and Handling

The ideal preparation maintains the tissue’s original structure and molecular composition, but challenges such as artifacts, distortions, and component loss can arise. Key steps include:

  • Prompt treatment of tissue samples post-removal to prevent autolysis (self-digestion by enzymes) and bacterial degradation.
  • Cutting tissues into smaller fragments to enhance fixative penetration and preservation.
  • Utilizing intravascular perfusion for certain organs or lab animals, which quickly distributes the fixative via the bloodstream.
  • Biopsy Needles are used for obtaining tissue samples from organs or lesions during minimally invasive procedures.
  1. 1. Removal of tissue: biopsy, necropsy.
  2. 2. Fixation is used: to terminate cell metabolism; prevent enzymatic degradation of cells and tissues by autolysis; kill pathogenic microorganisms; harden the tissue as a result of cross‐linking or denaturing proteins.
  3. A. Chemical fixatives:
  4. a. Simple and complex fixatives.
  5. b. Immersion and perfusion.
  6. B. Physical fixation – freezing.
  7. 3. Rinsing.
  8. 4. Dehydration 50 %, 70%, 90 %, 100 % and 100% ethanol.
  9. 5. Clearing in xylen.
  10. 6. Infiltration with melted paraffin at 56°C. 7. Embedding in paraffin – Edwin Klebs (1869).
  11. 8. Sectioning – microtome.
  12. 9. Staining: types of stains.
  13. 10. Mounting

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