To gram stain, an investigator smears a sample of bacteria on a slide, soaks it in a violet dye and then treats it with iodine. The slide is then rinsed with alcohol and counterstained with a pink dye called safranine.
The cell walls of gram-negative bacteria have a very low affinity for the violet stain, which is rinsed out by the alcohol. Once counterstained with safranine, however, the gram-negative bacteria appear bright pink to red. Gram-positive cell walls have a high affinity for the violet stain, and retain it even through the alcohol rinse. When the process is complete, they appear dark purple to brown.
Gram-staining is a 4 steps which uses certain dyes to make a bacterial cell stand out against against its background.
What things required
The specimen should be mounted and fixed on a slide before you proceed to stain it.
The reagents you will need are:
Before starting, make sure that all reagents, as well as the squirt-bottle of water, are easily accessible because you won't have time to go get them during the staining procedure.
Also, make sure you are doing this near a sink because it can get really messy.
Wear a lab coat.
1: Place your slide on a slide holder or a rack. Flood (cover completely) the entire slide with crystal violet. Let the crystal violet stand for about 60 seconds. When the time has elapsed, wash your slide for 5 seconds with water. The specimen should appear blue-violet when observed with the naked eye.
2: Now, flood your slide with the iodine solution. Let it stand about a minute as well. When time has expired, rinse the slide with water for 5 seconds and immediately proceed to step three. At this point, the specimen should still be blue-violet.
3: This step involves addition of the decolorizer, ethanol. Step 3 is somewhat subjective because using too much decolorizer could result in a false Gram (-) result. Likewise, not using enough decolorizer may yield a false Gram (+) results. To be safe, add the ethanol dropwise until the blue-violet color is no longer emitted from your specimen. As in the previous steps, rinse with the water for 5 seconds.
4: The final step involves applying the counter stain, safranin. Flood the slide with the dye as you did in steps 1 and 2. Let this stand for about a minute to allow the bacteria to incorporate the saffranin. Gram positive cells will incorporate little or no counter stain and will remain blue-violet in appearance. Gram negative bacteria, however, take on a pink color and are easily distinguishable from the Gram positives. Again, rinse with water for 5 seconds to remove any excess of dye.
After you have completed steps 1 through 4, you should blot the slide gently with bibulous paper or allow it to air dry before viewing it under the microscope.