The Gram Stain

The first staining procedure we did for Microbiology lab this semester dealt with the Gram Stain. The stain was, “…named after the Danish bacteriologist who originally devised it in 1882,”is done in order to allow for the classification of bacteria as either gram positive or gram negative, often for diagnostic purposes. The procedure consists of the following basic steps: a drop of primary stain (crystal violet) is applied to a heat fixed smear of the bacteria of interest, leaving all cells stained purple initially. Secondly, the bacteria are washed using distilled water and a single drop of IKI or Gram’s iodine is then applied over the smear so that CV-I complex crystals may be allowed to form. Next, after another washing with distilled water, ethyl alcohol or the decolorizing agent is applied so that the thin outer layer of gram negative bacterial cells is dissolved away, allowing for the loss of the primary stain in this particular type of organism (while the thickly walled gram positive bacteria are capable of retention of the CV-I complex). Lastly, after washing the decolorizing agent off with distilled water, the counterstain, Safranin, is applied so that the gram negative bacteria may be identified or colored by this reddish-pink dye. Therefore, when viewed microscopically, the gram negative cells are expected to appear pink in color (retain the counterstain color, Safranin) since the CV-I complex typically would have been washed out when treated with ethyl alcohol. However, because the cell structure of gram positive cells consists of a much thicker peptidoglycan layer (which allows for greater retention of the CV-I complex), those bacteria which are gram positive are expected to appear purple in color when viewed microscopically.