This overview of slide staining originally appeared on wardsci.com.
The exact chemistry involved in most staining procedures is unknown. There is probably more than one type of chemical interaction occurring during any staining procedure. Interactions can be electrostatic, covalent, or based on physical properties that affect stain penetration, including texture, density and solubility.
One prevalent mechanism is thought to be the electrostatic interaction of the chromogen with tissue molecules leading to the classification of many stains as acidophilic or basophilic. Basic dyes color acidic molecules - for example, hematoxylin, stains DNA (an acid) dark blue. Acid dyes color more basic molecules - for example, eosin, makes most cytoplasms red/pink. Continue reading ‘Slide Stain Guide’
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This overview of slide preparations originally appeared on wardsci.com.
Choosing the Type of Preparation
Even if you know what type of specimen you would like to examine, there are a variety of ways to prepare the specimen for the finished microscope slide. Our catalog describes specimen preparation in the following general categories:
- Wholemount - An entire specimen, such as an insect, is embedded in mounting resin directly on the slide and covered with a glass coverslip.
- Smear or Drop - The specimen(s) are in suspension then dried directly onto the glass slide where they are fixed, stained, and mounted in resin under a coverslip. This preparation is usually used for bacteria culture preparations or blood cells.
- Squash - The cell specimen is broken using pressure — usually used to release chromosomes from nuclei, then processed as a smear is.
- Section - A thin piece of specimen is shaved from the whole specimen to permit light to reveal greater structural detail. Sections are usually between 10 and 100 microns thick, which is usually thicker than one cell diameter. Therefore, several layers of cells may be present in the section.
- Thin section - In this case, sections are on the order of 1 micron thick, which is usually less than one cell diameter. Therefore, subcellular structures are more easily discerned than in thicker sections.
Types of Sections - Orientation
What is seen in a tissue section is often greatly dependent upon the plane of section or the direction the slice was made in. The most basic sections are cross section and longitudinal section. If you picture an object that is longer than it is wide, like a tree trunk, a cross section takes a section perpendicular to the long axis resulting in a slice that is relatively small and round. A longitudinal section would be parallel to the long axis and look like a long rectangle or oval. If that section was through the middle, such that you had a thin layer of bark visible on two opposite sides of the section, that would be a medial longitudinal section. If the sections were very close the edge of the tree trunk, the rectangle would have mostly bark and would be considered more tangential longitudinal sections.
Typical Section Preparation
The steps in making a section preparation are as follows:
- Collection and/or dissection of the specimen into a piece that will fit on a glass slide.
- Fixation: This step preserves the structure of the material, often by denaturing proteins. Formaldehyde is a common fixative that causes covalent cross-linkages to be added between proteins.
- Embedding: This step allows a supportive substance to infiltrate a specimen followed by hardening. Paraffin (wax) is commonly used to fill in spaces in tissue while it is in the liquid state (warm), then the entire block solidifies at room temperature. This permits the original shape of the specimen to be maintained through subsequent processing. Plastic is often substituted for paraffin.
- Sectioning: The supported specimen is cut into thin slices. This is usually done on a sharp metal blade mounted in a microtome to ensure that sections are of a known and constant thickness, usually around 25 microns.
- Affixing: The section is flattened onto a glass slide to permit it to adhere to the glass. This often requires a type of ?glue? to keep the section on the glass through further processing. Glass is used in making permanent slides for a variety of reasons. These include the fact that glass resists scratching, it can be cleaned better than plastic, and the optical properties of glass allow for better resolution than plastic.
- Staining: The slide with the affixed section is placed in an appropriate stain that will result in visual contrast between structures in the finished slide (see staining page). This step is often preceded by removal of wax from the slide and specimen.
- Dehydration: Water is removed from the tissue by repeated bathing in ethanol and other solvents. This makes the specimen more transparent so it can be viewed with greater clarity.
- Coverslipping: A mounting medium with optical properties similar to glass is used to permanently embed the specimen in resin and to permanently adhere the glass coverslip.
Different types of specimen preparations use a subset of the above steps, often in a different order, to make the finished slide.
Related Articles: Also see our Slide Stain Guide.
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