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 1,2.
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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 2:
- 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 3.