Gram Staining : Principle & Machanism, Steps, Disadvantages and Factors affecting gram Staining

Gram staining is a technique used to differentiate two major groups of bacteria based on their cell wall composition called gram positive and gram negative bacteria.

The technique was named after Hans Christian Gram who developed this method in 1884.
Gram staining is a preliminary test in the bacterial identification process. It plays an important role in clinical microbiology. It helps the medical professionals in the diagnosis of infectious diseases directly from the clinical sample.

The cell wall of gram positive bacteria is completely different from the gram negative bacteria. It is important for them to understand the Gram nature to provide the appropriate treatment for the infection.

Requirements of Gram Staining

  • Clean grease free glass slide
  • Nichrome wire loop
  • Bacterial cell suspension
  • 0.5% Crystal violet
  • Gram's lodine
  • 95% Ethanol
  • 0.5% Safranine/basic fuchsin

Steps of Gram Staining

Imagine our sample has a mixed culture with gram positive cocci and gram negative rods

Step-1 (Smear Preparation)

  • Prepare a thin smear of culture on a clean glass slide (oil free)
  • Allow to air-dry.

Step-2 (Heat Fixation)

  • Heat fix by exposing the smear to indirect flame
  • Heat fix slides above burner by moving slides through the peak of the flame.
  • Never heat fix wet slides !
  • Purpose of heat fixing is to kill and immobilize bacteria on slide.

Step-3 (Primary stain)

  • Add a few drops of Crystal violet on the smear.
  • Wait for a minute.
  • Wash the Crystal violet with water.
  • At this stage, all cells appear in purple colour.

Step-4 (Mordant)

  • Add plenty of Gram's lodine to the smear.
  • Wait for 1 minute.
  • Rinse with water.
  • Cells appear to be purple yet.

Step-5 (Decolourising)

  • Decolourise with 95% Ethyl Alcohol for not more than 30 seconds.
  • Wash with water.
  • At this stage, gram positive cells appear in purple colour And gram negative appear as colourless.

Step-6 (Counter stain)

  • Add counter stain Safranin to the smear.
  • Wait for 1 minute.
  • Wash with water.

Step-7 (Microscopic Observation)

  • Dry the slide and put a drop of immersion oil on the smear & Observe under 100X lens.

  • Gram positive cells retain the purple colour
  • Gram negative cells appear in pink colour.

Machanism of Gram Staining (Principle) :

The widely accepted theory is based on the differences in the cell wall composition between the gram-positive and gram-negative bacteria.

When crystal violet and iodine are added to the smear both will penetrate through the cell wall. And form a large crystal violet iodine complex (CVI complex) within the inner and outer layers of gram positive and gram negative bacteria.

The cell wall of gram-negative bacteria is thin and made of one or two layers of peptidoglycan. In addition to this it has got an outer lipopolysaccharide layer surrounding the cell wall.

  When a decolorizer like alcohol or acetone is added. The outer lipopolysaccharide layer will be completely dissolved leaving the thin peptidoglycan layer exposed.
The effective alcohol makes the peptidoglycan layer become perforated.

  In the decolorization step the gram-negative bacteria failed to retain the CVI complex and become colourless as the complex is washed away.

On contrary to this thick and multi-layered peptidoglycan in gram positive bacteria will be dehydrated by the addition of alcohol. Because of the thick peptidoglycan layer and dehydration by the alcohol treatment the CVI complex gets trapped in the cell.

  Therefore after the decolorization step the gram positive bacteria appear purple in color and the gram negative bacteria become colorless in the last step.

  When a counter stain likes a safranine is added the gram negative bacteria easily absorbed the stain and gives pink color to the cells. The gram positive bacteria remain purple in color due to the retention of CVI complex which is darker than Safranine.

Factors affecting Gram staining

  • Age of culture.
  • Excessive heat fixation.
  • Thick smear or overcrowding of cells.
  • Old staining reagents.
  • Air drying.
  • Over decolourization.

Disadvantages of Gram Staining

Athough the gram staining is used as primary test in the identification process this method will not be able to identify the bacteria to the species level. It will be used in combination of other modern and traditional identification tests
  The CVI complex may get lost from the gram positive bacteria due to over decolorization, which might lead to misinterpretation.
  There were evidences where some old gram positive cultures were not able to retain purple color and therefore observe as gram negative. 

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