Gram Staining

Gram Staining


Gram stain was originally devised by Dr. Christian Gram, a histopathologist, to stain bacteria in tissue sample. Now Gram stains is used widely to identify bacteria and an important part of microbiology and bacteriology.

Gram staining is not only used for identification of bacteria, but it also help to understand structure of bacteria, their growth requirements, susceptibility to antibiotics and pathogenicity.


Exact mechanism of gram reaction is not fully understood yet, but several suggestion are as follows

  • Gram positive cell have more acidic cytoplasm, which retain the basic primary dye more strongly than Gram negative bacteria, such as crystal violet.
  • The peptidoglycan layer in Gram positive bacteria are thick and thus retain dye-iodine complex more strongly than Gram negative bacteria, as Gram negative bacterial cell wall is more permeable.
  • High lipid content in Gram negative bacteria make them permeable to secondary dye after decolourization with organic solvent, like Acetone.


  • Primary staining is done with a para rosaniline dye, such as crystal violet, methyl violet or gention violet.
  • Application of a dilute solution of Iodine.
  • Decolorization with a organic solvent like, ethanol, acetone or aniline.
  • Counterstaining with another dye with contrasting colour such as carbol fuchsin, safranin or neutral red.


  • Crystal violet stain ( gention violet or methyle violet can be used)
  • Lugol’s iodine
  • Acetone-alcohol decolourizer ( Ethanol 95% v/v can be used, some prefer to use Acetone or ethanol-iodine solution. But acetone-alcohol is preferable, as it decolourize more rapidly and do not over decolourize like acetone)
  • Neutral red 1g/l (0.1% v/v)[ neutral re is preferable because it stains well gonococci and meningococci, but safranin can also be used. Dilute carbol fuchsin (1in10) is recommended for staining Vincent’s organism, Yersinia, Haemophilus, Compylobacter and vibroi species]


  1. Fix the smears. When gonococcus or meningococcus like intracellular organisms are suspected, fix with methanol for 2 min to avoid damaging pus cell.
  2. Cover the fixed smear with crystal violet stain for 30-60 seconds.
  3. Rapidly wash off the stain with clean water. ( If tap water is not clean, use filtered water or boiled rain water)
  4. Tip off all the water, pour Lugol’s iodine covering the smears and wait for 30-60 seconds.
  5. Wash off the iodine with clean water.
  6. Decolourize the smears rapidly with acetone-alcohol for few seconds. ( acetone-alcohol is highly inflammable, use with caution)
  7. Wash immediately with clean water.
  8. Cover the smears with neutral red for 2 Minuits.
  9. Wash off the dye with clean water.
  10. Place the slide in a draining rack to air dry.
  11. Examine under microscope – 1st with 40x objective to check the staining, then with 100x oil immersion for identification of bacteria and cell.


  • Gram positive bacteria   :               Dark purple
  • Yeast  cell                            :               Dark purple
  • Gram negative bacteria :               pale to dark red
  • Nucleus of pus cell           :               Red
  • Epithelial cell                      :               Pale red              


  • Report number of bacteria present in terms of Many/ Moderate/ Few/ Scanty
  • Gram reaction of the bacteria- weather gram positive or gram negative.
  • Morphology of bacteria: weather cocci, diplococcic, coccobacilli, bacilli, streptococci etc.
  • Weather the organism are intracellular/ not.
  • Presence and number of pus cells.
  • Presence of yeast cell and epithelial cells.
  • Example of report- “moderate numbers gram negative intracellular diplococcic and many pus cells” in case of urethral discharge.


Gram positive organism may lose their ability to retain the dye and may stain like gram negative in the following occasions

  • Cell wall damage due to antibiotic therapy or excessive heat fixation of the smears.
  • Over decolourization of the smears.
  • Use of iodine solution which is too old ie, yellow instead of brown in colour. So, keep iodine always in brown glass or other light opaque container.
  • Smears has been prepeared from an old culture.

Gram negative smears may look like gram positive in case of thick smears and if not fully decolourised.


Gram positive cocci

  • Staphylococcus
  • Stereptococcus
    • Alpha haemolytic: Pyogens, agalactiae
    • Beta haemolytic : Enterococcus
    • Gamma haemolytic: pneumonia, vitidans

Gram positive bacillus

  • Corynebacterium
  • Clostridium
  • listeria
  • Bacillus

Gram negative cocci

  • Neisseria gonorrhoeae
  • Neisseria meningitidis
  • Moraxella catarrhalis
  • Haemophilus influenza

Garm negative bacilli

  • Klebsiella pneumonia
  • Legionella pneumophila
  • Pseudomonas aeruginosa
  • E. Coli
  • Proteus mirabilis
  • Enterobacter
  • Serratia
  • Helicobacter pylori
  • Salmonella enteritidis
  • Salmonella typhi
  • Acenitobacter baumanni –associated with hospital acquired infection.


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