The Gram staining method was developed by Hans Christian Gram to differentiate between bacterial species. It divides bacteria into two groups - Gram positive and Gram negative - based on differences in their cell wall structure that determine whether they retain or release a purple crystal violet dye. Gram positive bacteria have a thick peptidoglycan cell wall that retains the dye, appearing purple under a microscope. Gram negative bacteria have a thinner peptidoglycan layer and an outer membrane, causing them to appear pink after a counterstain is applied. The Gram staining procedure involves applying and rinsing with crystal violet, iodine, decolorizer, and safranin solutions to visualize these differences between bacterial cell wall structures.

1. Study of bacteria by using
gram staining technique

2. The Gram staining method is named after the
Danish bacteriologist Hans Christian Joachim
Gram who used it to discriminate between
Pneumococci and Klebsiella pneumoniae
bacteria in lung tissue. It is a differential
staining method of differentiating bacterial
species into two large groups (Gram-positive
and Gram-negative) based on the chemical
and physical properties of their cell walls.

3. Bacterial species with walls containing small amounts of
peptidoglycan and characteristically,
lipopolysaccharide, are Gram-negative whereas
bacteria with walls containing relatively large amounts
of peptidoglycan and no lipopolysaccharide are Gram-
positive. Peptidoglycans are mainly a polysaccharide
composed of two subunits called N-acetyl glucosamine
and N-acetyl muramic acid.
The Gram-negative bacteria also have an additional outer
membrane which contains lipids, which is separated
from the cell wall by means of a periplasmic space

4. Gram-positive bacteria have a thick mesh-like
cell wall made of peptidoglycan (50–90% of
cell envelope), and as a result are stained
purple by crystal violet, whereas gram -
negative bacteria have a thinner layer (10% of
cell envelope), so do not retain the purple
stain and are counter-stained pink by the
Safranin.
Gram staining is thus based on the ability of
bacterial cell wall to retain the crystal violet
dye during solvent treatment by
peptidoglycan in the cell wall.

5. Gram staining consists of four components
• Primary stain (Crystal violet, methyl violet or
Gentian violet)
• Mordant (Gram's Iodine)
• Decolorizer (acetone/alcohol)
• Counterstain (safranin)

6. Material Required
Curd solution for bacteria (for gram positive bacteria,
Lactobacillus),
Half cooked rice solution (rich in gram negative bacteria,
Escherichia coli),
distilled water (preferably in a wash bottle),
Crystal Violet (primary stain: 1g crystal violet in 100 ml of distilled
water),
Gram's Iodine (mordant: 1g iodine and 2g Potassium iodide (KI) in
300 ml of distilled water),
Decolorizer (e.g. acetone or 95% ethanol),
Safranin (secondary stain: 1g safranin in 100 ml of distilled water),
Glass slides, Collecting Loop, Matchbox, Bunsen Burner, Slides,
Diamond marker, Coplin jars, Slide holder, blotting paper,
microscope.

7. Procedure:
• Mark the clean slides for different bacterial samples
(curd and half cooked rice solution).
• Put a small drop of the bacterial sample on one edge
of the slide and make a smear with another slide at
45o angle to the drop.
• For fixing the bacteria, heat the slide by passing the
slide with smear briefly through the spirit lamp
without exposing the dried film directly to the flame.
• Add crystal violet solution on the slide so as to flood
it for 60 sec.
• Remove the excess stain by washing the slide with
distilled water.

8. • Add Gram's Iodine solution to cover the smear on
the slide for 30 sec
• Wash the smear with distilled water and drain.
• Again wash the smear with acetone for
decolorization very briefly (2-3 sec or one dip).
• Wash the smear immediately with distilled water
to stop the decolorization.
• Counter stain the smear with safranin for 60 sec.
• Wash the smear on slide again with distilled
water and drain.
• Allow the smear to air dry and observe under a
microscope (40X)

9. Applications of Gram staining:
• Differentiation of bacteria into Gram positive and
Gram negative is the first step towards classification
of bacteria.
• It can be used as identification of bacteria in cultures.
• Observation of bacteria in clinical specimens
provides a vital clue in the diagnosis of infectious
diseases.
• Useful in estimation of total count of bacteria.
• Empirical choice of antibiotics can be made on the
basis of Gram stain’s report.
• Choice of culture media for inoculation can be made
empirically based on Gram’s stain report.

10. Precautions:
• Smear should be even and thin.
• The entire slide should not be covered with the
sample. This will make handling difficult and areas may
be missed during decolorization.
• Overheating should be avoided as it may alter cell
morphology or cause organisms to decolorize more
quickly.
• 95% ethanol will decolorize slower than
acetone/alcohol, than does acetone. Therefore, timing
for decolorization should be set accordingly and taken
care of.
• The decolorization step must be performed carefully,
otherwise over-decolorization may occur. This step is
critical and must be timed correctly otherwise the
crystal violet stain will be removed from the Gram-
positive cells also.