Understanding the Gram Stain of Hans Christian Gram

Gram staining is a method used in the medical laboratory to differentiate between two major groups of bacteria (Gram positive and Gram negative) based on the properties of their cell walls. The Gram stain primarily flags the thick layer of peptioglycan certain bacterial organisms which are thus identified as gram positive. The Gram Stain was initially created by Hans Christian Gram to differentiate between pneumococci and klebsiella pneumonia. Currently, gram stain is utilized in the lab as the first step conducted to identify bacterial organisms.


There are four basic components that are used while conducting a gram staining procedure: primary stain (primary stain can be methyl violet, Gentian violet and crystal violet); Mordant (usually Gram’s iodine); Decolourizer (can be acetone, ethyl alcohol or a mixture of ethanol and acetone); a Counterstain (can be safranin, neutral red or carbol fuchsin).


A blood smear is made on a glass slide in preparation for gram staining.  The smear is then flooded with one of the primary stains mentioned.  The primary stain makes all the bacteria on the slide appear violet.  The slide is left standing for one minute and then washed in water.

Two drops of Gram’s iodine are added to the smear and left to react for approximately one minute to form a complex composed of the dye and iodine in the cytoplasm of the bacterial cells. Gram’s iodine functions as a mordant.

The slide is washed again and then decolorized using acetone or ethyl alcohol.  A mixture of the two agents can also be used to decolorize the slide. Normally, decolorization should not exceed 30 seconds or 10 seconds when using acetone alone.  Caution should be taken not to over-decolorize or under-decolorize.

The smear should be washed again in water immediately after decolorizing followed by the addition of a few drops of a counterstain (neutral red, carbol fuchsin or safranin). After that a final wash is carried out and then the slide is dried and fixed using heat before being examined microscopically.


As described earlier, gram staining is basically used to differentiate between gram positive and gram negative bacteria. The staining technique has been widely used in medical diagnosis in the past but is currently being substituted by the more accurate molecular techniques.  Gram staining is, however, very effective in the diagnosis of certain conditions such as gonorrhea in which it achieves a specificity that is comparative to molecular techniques.  There are certain bacterial organisms that are neither gram positive or gram negative and therefore cannot be investigated using the gram technique.