Gram Staining

The Gram stain procedure is a differential staining procedure that

involves multiple steps. It was developed by Danish microbiologist Hans
Christian Gram in 1884 as an effective method to distinguish between
bacteria with different types of cell walls, and even today it remains one of
the most frequently used staining techniques. The steps of the Gram stain
procedure are listed below and illustrated in Table 1.

1. First, crystal violet, a primary stain, is applied to a heat-fixed smear,

giving all of the cells a purple color.
2. Next, Gram’s iodine, a mordant, is added. A mordant is a substance
used to set or stabilize stains or dyes; in this case, Gram’s iodine acts
like a trapping agent that complexes with the crystal violet, making the
crystal violet–iodine complex clump and stay contained in thick layers
of peptidoglycan in the cell walls.
3. Next, a decolorizing agent is added, usually ethanol or an
acetone/ethanol solution. Cells that have thick peptidoglycan layers in
their cell walls are much less affected by the decolorizing agent; they
generally retain the crystal violet dye and remain purple. However, the
decolorizing agent more easily washes the dye out of cells with thinner
peptidoglycan layers, making them again colorless.
4. Finally, a secondary counterstain, usually safranin, is added. This
stains the decolorized cells pink and is less noticeable in the cells that
still contain the crystal violet dye.

Gram-staining is a differential staining technique that uses a primary stain

and a secondary counterstain to distinguish between gram-positive and
gram-negative bacteria.

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Table 1. Gram stain process

Gram staining stems Cell effects Gram-positive

Step 1: Crystal Violet

Stains cells purple or blue.
primary stain added to the specimen smear

Step 2: Iodine
Cells remain purple or blue.
mordant, makes the dye less soluble so it
adheres to cell walls.

Step 3: Alcohol
Gram-positive cells remain purple or blue,
the decolorizer, washes away stain from Gram-negative cells are colorless.
gram-negative cell walls

Step 4: Safranin
Gram-positive cells remain a purple or blue.
counterstain allows dye adherence to gram- Gram-negative cells appear pink or red
negative cells

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Gram negative and gram positive organisms are distinguished from each
other by differences in their cell walls. These differences affect many
aspects of the cell, including the way the cell takes up and retains stains.

Gram positive cells take up the crystal violet, which is then fixed in the cell
with the iodine mordant. This forms a crystal-violet iodine complex which
remains in the cell even after decolorizing. It is thought that this happens
because the cell walls of gram positive organisms include a thick layer of
protein-sugar complexes called peptidoglycans. This layer makes up 60-
90% of the gram positive cell wall. Decolorizing the cell causes this thick
cell wall to dehydrate and shrink, which closes the pores in the cell wall and
prevents the stain from exiting the cell. At the end of the gram staining
procedure, gram positive cells will be stained a purplish-blue color.

Gram negative cells also take up crystal violet, and the iodine forms a
crystal violet-iodine complex in the cells as it did in the gram positive cells.
However, the cell walls of gram negative organisms do not retain this
complex when decolorized. Peptidoglycans are present in the cell walls of
gram negative organisms, but they only comprise 10-20% of the cell
wall. Gram negative cells also have an outer layer which gram positive
organisms do not have; this layer is made up of lipids, polysaccharides,
and proteins. Exposing gram negative cells to the decolorizer dissolves the
lipids in the cell walls, which allows the crystal violet-iodine complex to
leach out of the cells. This allows the cells to subsequently be stained with
safranin. At the end of the gram staining procedure, gram negative cells
will be stained a reddish-pink color.