Gram Stain Protocols
Ann C. Smith, Marise A. Hussey
ASM

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The Gram stain was first used in 1884 by Hans Christian Gram (Gram,1884). Gram was searching for a method that would allow visualization of cocci in tissue sections of lungs of those who had died of pneumonia. Already available was a staining method designed by Robert Koch for visualizing turbercle bacilli. Gram devised his method that used Crystal Violet (Gentian Violet) as the primary stain, an iodine solution as a mordant followed by treatment with ethanol as a decolorizer. This staining procedure left the nuclei of eukaryotic cells in tissue samples unstained while the cocci found in the lungs of those who had succumbed to pneumonia were stained blue/violet. Gram found that his stain worked for visualizing a series of bacteria associated with disease such as the “cocci of suppurative arthritis following scarlet fever”. He found however that Typhoid bacilli were easily decolorized after the treatment with crystal violet and iodine, when ethanol was added. We now know that those organisms that stained blue/violet with Gram’s stain are grampositive bacteria and include Streptococcus pneumoniae (found in the lungs of those with pneumonia) and Streptococcus pyogenes (from patients with Scarlet fever) while those that were decolorized are gramnegativebacteria such as the Salmonella Typhi that is associated with Typhoid fever.

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