Gram staining


Gram staining is a so called differential staining techniques, since one can distinguish two major groups of bacteria by this method. These two groups are gram positive and gram negative bacteria, which are stained purple and pink to red, respectively.


Gram-positive bacteria have a thick cell wall (peptidoglycan), which consists of several layers and can be likened to a network. Gram negative bacteria have a much thinner cell wall and also an outer membrane. Crystal violet (CV+), which is the primary dye binds to the negatively charged groups on the bacteria and stain them purple. Then iodine (I-) will be used to form a large complex (CV-I) with CV and thereby bind the stain to the bacterium. When Gram-positive bacteria are treated with the decolourizing solution (ethanol-acetone), the bacteria will be dehydrated and the colour retained. When Gram-negative bacteria are treated with the decolourizing solution the outer membrane will be dissolved and the thin peptidoglycan exposed, so that the CV-I complex is washed out. Then a counterstaining with safranin or basic fuchsin is performed to stain gram negative bacteria pink or red.


  1. Divide the slide with the help of the diamond pen.
  2. Disperse some colony material in a drop of NaCl, air dry (in the incubator, if necessary).
  3. Fix the specimen by moving the slide, with the preparation side up, 6-8 times through the burner flame.
  4. Add chrystal violet, wait for 1 minute.
  5. Flood gently with Lugol's solution.
  6. Add Lugol's solution (which contains iodine), wait for 1 minute.
  7. Flood gently with acetone-ethanol solution.
  8. Flood gently with water.
  9. Add safranin, wait for 20 seconds.
  10. Flood gently with water.
  11. Remove the excess of fluid with a paper towel and allow to air dry until the specimen is completely dry.

Gram-positiva bacteria

Members of the phyla Bacillota and Actinomycetota (exception: genus Mycobacterium).

Gram-negativa bacteria

Members of the phyla Bacteroidota, Campylobacterota, Cyanobacteria, Fusobacteriota, Pseudomonadota (exception: some members of the order Rickettsiales), Spirochaetota and Thermodesulfobacteriota.