Gram staining is a four part procedure which uses certain dyes to make a bacterial cell stand out against its background. The specimen should be mounted and fixed on a slide before you proceed and stain it. The reagents you will need to successfully perform this operation are:
- Crystal violet ( the primary stain)
- Iodine solution (the mordant)
- Decolorizer (ethanol is a good choice)
- Safranin (the counterstain)
- Water (preferably in a squirt bottle)
Before starting, make sure that all reagents, as well as the squirt bottle of water, are accessible. Do this near a sink and wear a lab coat.
- 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 a naked eye.
- Now, flood your slide with iodine solution. Let it stand for about a minute as well. Afterwards, rinse the slide with water for 5 seconds and immediately proceed to step 3. at this point, the specimen should still be blue-violet.
- This step involves addition of the decolorizer, ethanol. This step is somewhat objective because using too much decolorizer could result in a false Gram (-) result. Likewise, not using enough decolorizer may yield a false Gram (+) result. 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 the slides with water for 5 seconds.
- The final step involves applying the counterstain, 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 safranin. Gam (+) cells will incorporate little or no counterstain and will remain blue-violet in appearance. Gram (-) bacteria, however, take on a pink color and are easily distinguishable from the Gram (+) ones. Again, rinse with water for 5 seconds to remove any excess dye.
After completing steps 1—4, blot the slides gently with bibulous paper or allow it to air dry before viewing it under the microscope. DO NOT RUB THE SMEAR!