Gram Staining

Gram Staining

1. Transfer a loopfull bacteria culture onto slide

   • Add a loopfull of water and spread the bacteria
     • Let the bacteria dry
       • Place slides onto hot plate to heat fix bacteria
         • Cover the smear with crystal violet dye and leave for 30-40 seconds
           • Remove excess stain by washing with distilled water
             • Cover the smear with Gram's iodine for 1 minute
               • Tilting the slide, run ethanol/acetone down the smear until the decolourising agent that runs down the smear no longer has a purple tinge
                 • Wash slide with distilled water to remove excess ethanol/acetone
                   • Add safranine onto smear for 20-30 seconds
                     • Rinse slide with distilled water to remove excess dye
                       • Dry the slide by blotting with filter paper
                     • Secondary stain/counterstain
                     • This dye will stain decolourised gram-negative bacteria
                 • Only gram-positive bacteria will retain the dye. This step will differentiate the bacteria.
                 • Overwashing may cause gram-positive bacteria to decolourise
               • Iodine combines with dye in the cell to crystal violet-iodine complex (CV-I)
           • Bacteria will absorb the dye
           • Primary stain
         • In order for bacteria to stick to the slide
     • To dilute the bacteria