The Use of Stains in Microscopy
Stains and the Prokaryotic Envelope
Prokaryotes are difficult to see in wet mounts because they are translucent. Wet mounts of live cells are useful in observing motility (swimming), the bacteria lack contrast against the white background. While special types of light microscopy have been invented to create better contrast stains are more frequently used to visualize cells in the lab. Stains or dyes colorize the cells or the background so they can be more clearly observed.
The prokaryotic cell wall and membrane is composed of phospholipid bilayers, proteins, peptidoglycan, lipopolysaccharides (in gram positive bacteria), and teichoic acids (in gram negative bacteria). Lipopolysaccharides and teichoic acids in particular carry negative charges and are responsible for giving the bacterial envelope an overall negative charge.
Stains or dyes used to colorize cells in microbiology are usually ionic compounds that disassociate into ions when mixed with water. The ion of the compound responsible for the color is known as the chromatophore. Stains are divided into basic and acidic stains based on the charge of the chromatophoric ion.
Basic Stains
Basic stains have positively charged chromatophores that are attracted to and bind to the negatively charged bacterial cell envelope. Since basic stains produce colorized cells against a white background (after rinsing), they are also called direct stains. Common basic stains used in microbiology labs include methylene blue, crystal violet, safranin, and carbofuschin.
Basic stains must be used with bacterial smears that are fixed to a microscope slide so that unbound stain can be rinsed away without fear of losing the specimen. The fixing of bacteria to the slide often involves heating or treatment with alcohol, both of which dehydrate and slightly distort the shape of the bacterial cells. For this reason, using basic stains may not be the best way to discern the cell morphology (shape).
Acidic Stains
Acidic stains have negatively charged chromatophores that are repelled by the bacterial cell envelope. They are usually mixed together with a small amount of liquid culture and spread or "painted" across a microscope slide. The slide is then viewed after the stain has dried to prevent getting stain on the microscope lenses.
Note that in preparing a negatively stained specimen, the cells are not distorted by heat or fixing chemicals. This makes the negative stain a better procedure for observing cell morphology.
Acidic stains produce clear, white bacterial cells against a dark field. This is the reverse of direct staining, which produces dark or colored cells against a white field. For this reason, acidic stains are also known as negative stains. The most common negative stains are india ink and nigrosin.
Staining Singly or in Combination
When a single stain is used to visualize cell morphology, the procedure is said to involve a simple stain. But staining procedures have also been designed to reveal differences in the characteristics between bacteria. Procedures which often involve several stains are called differential stains. Differential staining procedures include the gram stain, acid-fast stain, endospore stain, and flagellar stain.