Atlas of Simple Stains, Simple Stains, , Bacterial Cellular Morphology and Simple Stains, A Photographic Atlas for the Microbiology Laboratory 4th edition 2011
Purpose
In Section 4 you were introduced to two of the three important features of a microscope and microscopy: magnification and resolution. A third feature is contrast. To be visible, the specimen must contrast with the background of the microscope field. Because cytoplasm is essentially transparent, viewing cells with the standard light microscope is difficult without stains to provide that
contrast. Once stained, cell morphology, size, and arrangement then may be determined.
Principle
Stains are solutions consisting of a solvent (usually water or ethanol) and a colored molecule (often a benzene derivative), the chromogen. The portion of the chromogen that gives it its color is the chromophore. A chromogen may have multiple chromophores, with each intensifying the color. The auxochrome is the charged portion of a chromogen and allows it to stain the cell through ionic or covalent bonds.
Basic stains (where the auxochrome becomes positively charged as a result of picking up a hydrogen ion or losing a hydroxide ion) are attracted to the negative charges on the surface of most bacterial cells. Thus, the cell becomes colored (Figure 5-1). Common basic stains include methylene blue, crystal violet, and safranin. Examples of basic stains may be seen in Figures 5-2 and 5-3, and in A Gallery of Bacterial Cell Diversity (pages 39–44).
5-1 CHEMISTRY OF BASIC STAINS Basic stains have a positively
charged chromogen (●), which forms an ionic bond with the negatively
charged bacterial cell, thus colorizing the cell.
5-2 A SIMPLE STAIN USING SAFRANIN
Safranin is a basic stain. Notice that the
stain is associated with the cells and not
the background. The organism is Klebsiella
mobilis (formerly Enterobacter aerogenes),
grown in culture. Cell dimensions are
0.3–1.0 µm wide by 0.6–6.0 µm long.
Basic stains are applied to bacterial smears that have been heat-fixed. Heat-fixing kills most of the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible. It also distorts the cells to some extent.
5-3 A SIMPLE STAIN USING CRYSTAL VIOLET
This micrograph shows Propionibacterium acnes
stained with the basic stain crystal violet. P. acnes is a commensal
living on the skin of most humans. It has been associated with the skin
condition acne. Cell dimensions are 0.5–0.8 µm wide by 1–5 µm long
Suggested Reading
- Michael J. Leboffe & Burton E. Pierce. A Photographic Atlas for the Microbiology Laboratory 4th edition 2011
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