If staining a clinical specimen, smear a very thin layer onto the slide, using a wooden stick. Do not use a cotton swab, if at all possible, as the cotton fibers may appear as artefacts. The smear should be thin enough to dry completely within a few seconds. Stain does not penetrate thickly applied specimens, making interpretation very difficult.
B. Spread the culture with an inoculation loop to an even thin film over a circle of 1.5 cm in diameter, approximately the size of a dime. Thus, a typical slide can simultaneously accommodate 3 to 4 small smears if more than one culture is to be examined.
C. Air-dry the culture and fix it or over a gentle flame, while moving the slide in a circular fashion to avoid localized overheating. The applied heat helps the cell adhesion on the glass slide to make possible the subsequent rinsing of the smear with water without a significant loss of the culture. Heat can also be applied to facilitate drying the the smear. However, ring patterns can form if heating is not uniform, e.g. taking the slide in and out of the flame.
2. Gram Staining:
A. Add crystal violet stain over the fixed culture. Let stand for 10 to 60 seconds; for thinly prepared slides, it is usually acceptable to pour the stain on and off immediately. Pour off the stain and gently rinse the excess stain with a stream of water from a faucet or a plastic water bottle. Note that the objective of this step is to wash off the stain, not the fixed culture.
B. Add the iodine solution on the smear, enough to...