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Name: Jignesh
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Age: 18
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When substances are stained, it is really due to a chemical reaction between the stain and something in the substance. Bacteria have a substance in their cell walls called peptidoglycan(PG)- a sugar-protein substance. Bacteria come in two types: those that have a lot of PG in their cells walls and those that have a small layer. A staining process called the Gram stain is used to stain bacteria. This is a differential stain because some bacteria are stained with one type of stain and the other type takes up another type of stain. The first stain is called crystal violet. After one minute it is rinsed off and iodine is added to help the stain stick to the bacteria. The alcohol is added. The bacteria that have lots of PG don't let go of the stain and they remain purple. We call them Gram positive. The other type that has only a little PG lets go of the purple stain and it all washes out. They then need to be stained again, but with a different stain. This stain is called safranin and is red. The bacteria that are red are said to be Gram negative.

Van Hoeck

The Gram stain is one of the most important stains in microbiology. It devides bacteria into Gram positive (single membrane) or negative (inner and outer membrane) and in this simple way a major morphological difference is made apparent. There is still debate about the actual mechanism behind the different colors observed in the stain. I suggest you read about Gram stains in the following four websites:

A simple description of the Gram stain from the University of Manchester at

Or have a look at

also contains a questionair, do you know your bacteria? The stain file ( ) gives the two different explanations of the mechanism behind the stain. The chemicals used in the stain are explained at
(American Chemical Soc.).

With this information your question is probably answered.

Dr. Trudy Wassenaar

It has been some time but as I remember red is the mordant which fixes the crystal violet into an insoluble complex. Gram Pos bacteria posess a monolayered cell wall (with park peptide linkages etc.) while the Gram neg are more complex in their cell wall with two structurally distinct layers (without a park peptide). Because of their cell wall structure the mordant does not fix the crystal violet on the gram neg so when they are destained with ethanol or acetone the crystal violet is washed away.

peter faletra

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