The gram stain permits the separation of all bacterial species into two large groups, those which retain the primary dye (gram-positive), and those which lose the primary dye and take the color of the secondary dye (gram-negative). The mechanism of gram stain is based on the distinctive chemistry and physical properties of the cell wall, possibly the lipid content. However, the exact mechanism of gram stain is still unknown.
Reagents

*The safranin counter stain may be substituted with basic fuchsin counter stain catalog # 435A for better Anaerobe staining.

Specimen Collection
Organisms being stained by the gram method are usually taken from a solid or liquid medium on (in) which they have been cultured from their original source (e.g. wounds, throat, swabs, sputum, etc.). An aqueous suspension is made, in the case of the solid medium, by taking a small amount of the material and suspending it in a drop of distilled water on a microscope slide. Care should be taken not to make the smear too thick. In the case of a liquid medium, a drop is used directly from the culture container. However, due to the solids from the medium, this method is not always satisfactory. The suspension made by either method is air dried, then “fixed” by passing rapidly through a Bunsen burner flame two or three times. Allow the smear to cool before staining.
Procedure

1. Place the “fixed” smear on a staining rack and cover completely with crystal violet for 30-60 seconds.
2. Wash off the stain with distilled water.
3. Cover the slide with iodine for 30 seconds.
4. Wash off with distilled water.
5. Decolorize with Gram decolorizer for 10-15 seconds.
6. Wash thoroughly with distilled water.
7. Cover completely with safranin for 30-60 seconds.
8. Wash with distilled water and air dry. Examine under immersion oil.

Note: Staining times may vary to suit the individual.

Sources of Error

1. Overheating (burning) during fixation can be avoided by just touching the back of the slide to the back of the hand each time the slide has been passed though the flame.
2. Do not stain smears which have only been air dried. Smears must also be “fixed”.
3. Smears should not be too thick. After air drying, examine under a microscope. If there are no areas of bacteria separation, more water should be added to dilute the smear.
4. After staining it is essential that the back surface of the slide is wiped clean.
5. If washing with distilled water is not done adequately, crystallization of the stain may appear on the slide.

Bibliography

1. Gram, C. Fosrtschr. Med. 2 185, 1884.