Gram Stain

Gram staining distinguishes cells based on bacterial cell walls.

Gram-positive organisms have a thick cell wall and outer membranes that retains crystal violet, while Gram-negative cells have no inter-glycine bridge and thin cell walls which eventually are stained pink. There are several potential problems with Gram staining: old Gram-positive cultures may stain Gram-negative; Gram-negative smears will not decolorize if the smear is too thick; samples may become over-decolorized and falsely appear Gram-negative; and Gram stains appear greenish under phase optics.

Step	Overview
Apply Culture	If staining a broth culture, use a sterile loop to transfer a small droplet onto a glass slide. If staining a growth from a solid medium, first place a small droplet of water onto a slide and then transfer some culture into the droplet using a sterile loop. Repeat this procedure onto different parts of the slide using a gram-positive control and a gram-negative control.
Air Dry & Fix	Afterward, allow the smears to air dry and then pass the slide thrice rapidly through a Bunsen burner flame (smear side up). If you cannot read print through the smears, then they are too thick. The slide should not be so hot that it hurts to touch.
Crystal Violet	Apply a solution of crystal violet to the heat-fixed smear and let soak for about one minute. This is also called the staining step.
Water Rinse	Rinse slide under gently flowing water. Shake off excess water.
Iodine	Squirt the slide with iodine to remove excess water, then apply iodine and let soak for about one minute.
Alcohol	Gently spill a few drops of 95% ethanol onto the slide, then tilt the slide back and forth. Repeat this step twice or thrice, until purple stain ceases to drain from the smears. The alcohol washes crystal violet from thin Gram-negative cell walls, but thick Gram-negative cell walls still retain the crystal violet. This is a critical step: if not enough alcohol is applied, then all cells will appear Gram-positive; if too much alcohol is applied, then all cells will appear Gram-negative.
Water Rinse	Rinse slide under gently flowing water. Shake off excess water.
Safranin	Squirt the slide with safranin to remove excess water, then apply safranin and let soak for about one minute. This step stains the washed-out Gram-negative cells pink, and is also called the counter-stain.
Water Rinse	Rinse slide under gently flowing water. Shake off excess water.
Blot & Dry	Gently blot the slide with a paper towel. Do not rub. Rapidly pass the slide once through the Bunsen burner flame.
Examine	Examine the slide using microscopy. Gram-stained slides can be studied using oil immersion. Make sure your Gram-positive control is purple, and that your Gram-negative control is pink. Compare your sample to these controls to conclude the Gram nature of the organism.