2.4 Staining microscopic specimens  (Page 5/11)

Capsule staining

Certain bacteria and yeasts have a protective outer structure called a capsule. Since the presence of a capsule is directly related to a microbe’s virulence (its ability to cause disease), the ability to determine whether cells in a sample have capsules is an important diagnostic tool. Capsules do not absorb most basic dyes; therefore, a negative staining technique (staining around the cells) is typically used for capsule staining . The dye stains the background but does not penetrate the capsules, which appear like halos around the borders of the cell. The specimen does not need to be heat-fixed prior to negative staining.

One common negative staining technique for identifying encapsulated yeast and bacteria is to add a few drops of India ink or nigrosin to a specimen. Other capsular stains can also be used to negatively stain encapsulated cells ( [link] ). Alternatively, positive and negative staining techniques can be combined to visualize capsules: The positive stain colors the body of the cell, and the negative stain colors the background but not the capsule, leaving halo around each cell.

Endospore staining

Endospores are structures produced within certain bacterial cells that allow them to survive harsh conditions. Gram staining alone cannot be used to visualize endospores, which appear clear when Gram-stained cells are viewed. Endospore staining uses two stains to differentiate endospores from the rest of the cell. The Schaeffer-Fulton method (the most commonly used endospore-staining technique) uses heat to push the primary stain ( malachite green ) into the endospore. Washing with water decolorizes the cell, but the endospore retains the green stain. The cell is then counterstained pink with safranin . The resulting image reveals the shape and location of endospores, if they are present. The green endospores will appear either within the pink vegetative cells or as separate from the pink cells altogether. If no endospores are present, then only the pink vegetative cells will be visible ( [link] ).

Endospore-staining techniques are important for identifying Bacillus and Clostridium , two genera of endospore-producing bacteria that contain clinically significant species. Among others, B. anthracis (which causes anthrax ) has been of particular interest because of concern that its spores could be used as a bioterrorism agent. C. difficile is a particularly important species responsible for the typically hospital-acquired infection known as “C. diff.”