Gram stain

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The Gram stain (or Gram method) is a key microbiological method for staining bacteria. The process relies upon two stains, the first, a complex of crystal violet and iodine, and the second, safranin, a red counterstain. The staining procedure subdivides bacteria into Gram-positive and Gram-negative bacteria, with important implications for their further categorization and treatment .

Gram staining procedure

It is a straightforward rapid staining method detailed in the reference. After each treatment step the slide is washed with water for 5 seconds to remove any excess reagent and excess water shaken off :

  • bacterial smear is heat-fixed to the glass slide
  • smear is initially covered with crystal violet for 30 seconds
  • slide washed
  • smear is treated with Gram's iodine solution for one minute
  • slide washed
  • decolourisation with 1:1 95% ethanol-acetone solution
  • slide washed
  • covered with counterstain, usually safranin O (in ethanol)
  • slide washed
  • smear is viewed with high-magnification oil immersion microscopy to decide if Gram positive or negative
    • Gram-positive bacteria are purple
    • Gram-negative bacteria are pink

    Mechanism of staining

    Fundamentally the method depends upon the difference in cell wall composition of the two types of bacteria. The cell walls of Gram-positive organisms are thick and retain the violet-iodine complex after treatment with alcohol retaining a purple hue. By contradistinction, in gram-negative organisms the cell wall (as it is) is much thinner and the initial dye is washed out by the decolourant, and the micro-organisms retain only the counterstain, rendering them pink (the counterstain attaches to the Gram-positive bacteria but cannot be seen due to being overwhelmed by the dark purple color of the crystal violet.

    History and etymology

    The Gram stain was developed by a Danish physician, Hans Christian Joachim Gram (1853-1938), whilst he studied methods to double stain renal histopathological specimens in Copenhagen in 1883. Serendipitously he found that some bacteria remained color-fast after being washed with alcohol .