A smear specimen for microscopic study is usually prepared by rolling a small quantity of the specimen material across a glass slide. A drop of saline in which the specimen has been emulsified can also be used. If the slide is also to be stained, it is generally fixed by rinsing in methanol. For direct examination of unstained material, phase-contrast microscopy can be used.

Smears are most often observed after they have been stained. Stains are salts composed of a positive and a negative ion, one of which is colored. Structures present in the specimen pick up the stain and make the organism visible under the light microscope. One staining procedure, called the negative stain, colors the background but leaves the organisms themselves uncolored. The gross structure of the organisms can then be studied.

Bacterial stains are of two major types: simple and differential. A simple stain consists of a coloring agent such as gentian violet, crystal violet, carbol-fuchsin, methylene blue, or safranin O. A thin smear of sampled organisms is stained and then observed under an oil immersion lens. A differential stain is one in which two chemically different stains are applied to the same smear. Organisms that are physiologically different pick up different stains.

The Gram stain is the most important of all bacteriologic differential stains. It divides bacteria into two physiologic groups: Gram-positive and Gram-negative organisms. The staining procedure consists of four major steps: (1) staining the smear with gentian or crystal violet; (2) washing off the violet stain and flooding the smear with an iodine solution; (3) washing off the iodine solution and flooding the smear with 95% alcohol or an acetone–alcohol mixture; and (4) counterstaining the smear with safranin O, a red dye. The Gram stain permits morphologic study of the sampled bacteria and divides all bacteria according to their ability or inability to pick up one or both of the stains. Gram-positive and Gram-negative bacteria exhibit different properties, which helps to identify and differentiate them.

Stains other than the Gram stain are used for examining bacteriologic smears. Some, such as the acid-fast stain, can identify organisms of the genus Mycobacterium. Other stains differentiate certain structures, such as capsules, endospores, or flagella.