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The True Gram Stain Mechanism

Author: Gabriel Ma

Introduced in 1882, the Gram staining technique is one of the most important techniques in microbiology. Discovered by the Danish bacteriologist Hans Christian Gram, it is used to distinguish between gram-positive and gram-negative bacteria.

Gram-negative cells have a cytoplasmic membrane (CM) on the inside, a peptidoglycan mesh (PM) above that, then an outer membrane (OM) above that. In these cells, the PM is very thin. Meanwhile, gram-positive cells also have a CM on the inside, but then a really thick PM above, with nothing above that. The periplasmic space is the area between each of these layers, regardless of gram type. For the sake of this article, components within each layer are not important. Cell membrane differences between bacterium allow the gram stain to take advantage of this difference by exposing the bacteria to different chemicals and noting how well it retains them.

Gram Staining Process

Bacteria are placed on a slide, and crystal violet (CV) dye is added for the first staining round. After adding iodine, which prevents easy removal of the dye, a decolorizer is used to attempt to remove the dye. Bacteria that retain the original crystal violet dye appear purple under a microscope and are G+ organisms. Then, a counterstain is added, which gives G- bacterium a pink colour so that they’re easier to identify.

Old vs New Beliefs

Prior to Michael J. Wilhelm et al.’s article revealing how gram stains work, it was believed that CV crosses the PM and CM to participate in equilibrium in the cytosol. However, experiments showed this to be false.

In reality, the CV and iodine are isolated in the PM. Since the PM of gram-positive bacteria is really thick, they remain intact after being exposed to the decolorizer. On the other hand, gram-negative bacteria lose their PM because it is thin and the decolorizer can also remove the OM.


There are no changes that need to be made to the gram staining process. All previous uses of the same staining process remain valid. The only change is our understanding of how it works.



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