Cytology · MU Sofia

2. Collection of material for microscopic examinations. Fixation.

Covers the preparation of tissue for microscopy, starting with collection and handling: prompt processing to prevent autolysis, cutting into fragments, intravascular perfusion, and biopsy needles. It then explains fixation's purpose and sorts methods into chemical, physical and heat fixation, comparing formalin, glutaraldehyde, osmium tetroxide and heavy-metal fixatives, with an image of tissue before and after formalin fixation.

Inside: 3 sections · 2 labelled pictures · 14 flashcards · 2 min

Topic 2 of 97 in the MU Sofia syllabus for Cytology (Medicine, Year 1). The free AceMed note covers it in 3 sections, about 303 words (2 min of reading), with 14 flashcards.

In this note

  1. Collection of material for microscopic examinations. Fixation.
  2. Slide Preparation for Microscopic Examination
  3. Fixation

Slide Preparation for Microscopic Examination

The preparation of slides for microscopic examination is a meticulous process that ensures the tissue’s structure and molecular integrity are preserved for accurate observation. This involves tissue collection, fixation, and subsequent steps such as embedding and sectioning.

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 could be needed For obtaining tissue samples from organs or lesions during minimally invasive procedures

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 andthe cellular components being examined.

This image shows Before formalin fixation and (b) after formalin fixation

Types of Fixation:

  1. 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.
  1. 2. Physical Fixation:
  • Methods such as freezing preserve tissue by rapid cooling, commonly used for histochemical studies where enzyme activity needs to be preserved.
  1. 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.

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