Ask A Scientist© Molecular Biology Archive

name        JIGNESH
status      student
age         18

Question -  WHY GRAM NEGATIVE BACTERIA ARE STAINED RED IN COLOR ?
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
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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
http://www.infection-control.man.ac.uk/gram.html Or
have a look at bartleby.com:
http://www.bartleby.com/65/gr/Gramssta.html
also contains a questionair, do you know your
bacteria?
The stain file
(http://members.pgonline.com/~bryand/theory/gram.htm )
gives the two different explanations of the mechanism
behind the stain.
The chemicals used in the stain are explained at
http://pubs.acs.org/hotartcl/cenear/960923/gram.html
(American Chemical Soc.).

With this information your question is probably
answered.

Dr. Trudy Wassenaar
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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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