P.O.

Box 5786
Ithaca, New York 14852-5786
t. 607 253-3900
f. 607 253-3943
w. diagcenter.vet.cornell.edu

GRAM STAIN PROTOCOL

This technique is used to stain a slide such as a fecal smear to observe the bacterial microflora
present based on their gram stain reaction.

• “Heat-fix” the slide with the specimen by passing it over a heat source, such as a
flame, several times using a clothes pin or forceps. The slide should be passed very
quickly through the flame and not be heated excessively. Place slide on the staining
tray.

• Flood the fixed smear with crystal violet solution (#1) and allow to remain for 1
minute.

• Rinse off the crystal violet with distilled or tap water.

• Flood the slide with iodine solution (#2). Allow to remain for one minute.

• Rinse off the iodine solution with distilled or tap water.

• Holding slide on a tilt with a clothes pin, flood the slide with decolorizer (#3) for one to
five seconds.

• Rinse off the decolorizer with distilled or tap water.

• Flood the slide with safranin (#4). Allow to remain for 30 seconds.

• Rinse off the safranin with distilled or tap water.

• Dry the slide on bibulous paper or absorbent paper and place in an upright position.

Microscopically examine the slide for bacterial organisms under a 100X objective. Observe
several fields on the slide for bacterial organisms. Describe the gram reaction of any
organisms seen. Gram-positive bacteria stain deep violet to blue and gram-negative bacteria
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stain pink to red. If your slide is all one color (either pink or blue), then the slide may either
have been over or under decolorized. Unless it is a gram stain of a pure culture, there
normally should be material staining both colors somewhere on the slide.

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