Biological sample preparation involves a series of crucial steps to preserve their structure and composition for analysis using techniques like light microscopy and electron microscopy (EM). The primary goal is to maintain the sample's integrity, preventing degradation and distortion.
Key Steps in Biological Sample Preparation
The conventional method focuses on three main steps:
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Fixation: This initial step stabilizes the sample's structure, preventing degradation from cellular processes like autolysis (self-digestion). Fixatives, such as formaldehyde, glutaraldehyde, or osmium tetroxide, cross-link proteins and other molecules, preserving the sample's three-dimensional structure. The choice of fixative depends on the specific application and the type of biological sample being prepared.
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Dehydration: Water is removed from the sample gradually through a series of increasing alcohol concentrations (e.g., ethanol or methanol). This process is essential because embedding media are typically non-polar and incompatible with water. Careful dehydration avoids shrinkage and distortion of the sample.
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Embedding: The dehydrated sample is then infiltrated with an embedding medium, such as paraffin wax (for light microscopy) or resin (for electron microscopy). This medium provides support and allows for thin sectioning of the sample. Paraffin wax is relatively soft, suitable for light microscopy, while resins are harder and provide the necessary rigidity for the thin sections required in electron microscopy.
Impacts of Preparation Methods
It's important to note that these preparation methods, while crucial, can impact the sample. As mentioned in the reference material, "These procedures result in increased permeability of the cell membrane with resultant loss or redistribution of mobile intracellular elements." This means that some components of the cell may be lost or their location altered during the preparation process. Therefore, researchers must be mindful of these limitations when interpreting their results.
Examples of Sample Types and Preparation Methods
- Tissue samples: Typically fixed, dehydrated, and embedded in paraffin or resin for sectioning.
- Cell cultures: Can be fixed directly on slides or in suspension for various imaging techniques.
- Blood samples: Prepared using specialized techniques, often involving centrifugation to separate components like serum and cells.
