Download gram negative bacteria will be red.

Laboratory Experiment
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The most used stain in bacteriology
developed in 1884 by Hans Christian Gram
This procedure is a differential stain method meaning that bacteria will give
different results depending on their cell wall chemistry.
The Gram stain divides bacteria into two groups based on their reaction to the
stain
Gram positive bacteria will be purple after the last step; gram negative
bacteria will be red.
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The four steps of the Gram stain
1. Primary stain - cover the smear with crystal violet for one minute. All bacteria will take
up this dye and appear purple. Rinse off the excess dye with water.
2. Mordant - Gram's iodine is added to interact for one minute with the crystal violet. This
complex will be difficult to remove from certain bacteria during the next (decolorization)
step. Excess iodine is rinsed off with water.
3. Decolorization - 95% ethanol is briefly (10-20 seconds) applied to the smear followed by
a water rinse.
4. Counterstain - Safranin is added for 20 seconds to dye any decolorized cells. It will not
change the color of the cells that retain the crystal violet.
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GRAM STAIN REACTIONS
STEP
TIME
GRAM +
GRAM -
Crystal violet
1 min
purple
purple
Gram's iodine
1 min
purple
purple
95% ethanol
20 sec
purple
colorless
Safranin
20 sec
purple
red
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PROCEDURE – VIDEO DEMO
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PRINCIPLE OF THE GRAM STAIN
The cell wall composition of Gram negative bacteria differs from that of Gram positives.
Since Gram negative bacteria have a high lipid content in their cell walls, one theory holds
that the decolorizer (acetone or ethanol) solubilizes the outer membrane of the cell wall thus
releasing the crystal violet. Therefore, after the decolorization step, Gram negative
organisms will appear colorless while Gram positives will appear purple. In the cell wall of
Gram positive organisms, the decolorizer is unable to act as a solvent thus the crystal violet
remains.
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REAGENTS AND MATERIALS
Crystal violet solution [primary stain]
(0.4% crystal violet in aqueous alcohol)
Iodine solution (stabilized) [mordant]
(13% polyvinylpyrrolidone-iodine complex in 1.9% KI)
Decolorizer solution [decolorizer]
(denatured ethyl alcohol:acetone, 3:1)
Safranin solution [counterstain]
(0.25% safranin in 20% ethyl alcohol)
Glass microscope slides
Immersion oil
Brightfield microscope
Bacterial culture(s):
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Limitations of the Gram Stain
A fresh culture should be used because old gram positive bacteria may decolorize and
stain with safranin.
Some organisms are gram variable, that is, some isolates may be gram positive, some
gram negative and some have cells staining with both characteristics.
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Examples…..
cocci
Neisseria gonorrhoeae
Staph. epidermidis
bacilli
S. aureus
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