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EXPERIMENT NO- 02
Gram Staining and Study of
morphology of bacterial cells ▪ Aim:
To Differentiate gram positive and gram negative using
Gram Staining methods.

▪ Objective:
1.To prepare smear from broth/plate culture.
2.To understand the technique and principle of gram stain.
To stain the smear using !ram stain.
4.To report the result of the unknown smear.

▪ Principle:
What is gram staining?
Gram Staining is the common, important, and most
used differential staining techniques in microbiology, which
was introduced by Danish Bacteriologist Hans Christian
Gram in 1884. This test differentiate the bacteria into Gram
Positive and Gram Negative Bacteria, which helps in the
classification and differentiations of microorganisms
How Gram staining is helpful. (Significant)
The main benefit of a gram stain is that it
helps your doctor learn if you have a bacterial
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infection, and it determines what type of

bacteria are causing it. This can help your
doctor determine an effective treatment plan
Difference Between the Gram positive and
Gram negative Bacteria ▪ Gram Positive
Bacteria:
The cell wall of gram-positive bacteria is composed of thick
layers peptidoglycan
In the gram staining procedure, gram-positive cells retain
the purple coloured stain.
Gram-positive bacteria produce exotoxins few

examples of gram-positive bacteria.

Gram-positive bacteria include the bacteria of genre

Staphylococcus, Streptococcus, Enterococcus. These
bacteria are the most common cause of clinical infections.

▪ Gram Negative Bacteria:

The cell wall of gram-negative bacteria is composed of thin
layers of peptidoglycan.
In the gram staining procedure, gram-negative cells do not
retain the purple coloured stain.
Gram-negative bacteria produce endotoxins Is it easier
to kill gram-positive bacteria?
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The cell wall of the gram-positive bacteria absorbs

antibiotics and cleaning products. Because of the outer
peptidoglycan layer, they are easier to kill.
Gram-negative bacteria cannot be killed easily.

What infections are caused by grampositive

bacteria?
Gram-positive bacteria usually cause Urinary Tract
Infections. These are caused commonly in people who are
more prone to urinary tract infections or are elderly or
pregnant.

Which infections are caused by

gramnegative bacteria?
The gram-negative bacteria cause various infections in
humans such as indigestion, food poisoning, pneumonia,
meningitis and other bacterial infections in the blood cells,
bloodstream, wound infections, etc. The infections are caused
by Acinetobacter, Pseudomonas aeruginosa and
E.coli.

▪ Material Required:
1.Light Microscope
2.Glass slide
3.Immersion Oil
4.Normal Saline
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5.wire Loop
6.Staphylococcus aureus plate culture
7.Electrical Incinerator
8.Gram stain reagent
i. primary stain(Crystal violet)
ii. Mordant (Gram's iodine)
iii. Decolourizer (acetone alcohol)
iv. Counterstain (Safranin)

Procedure:
• Take a glass slide and rub the alcoholic cotton on that
slide. After that keep the glass to dry.
• After drying of slide, we take a curd sample. We will
take nichrome loop in the beaker and move that.
• Now, in the next step we will go through the heat
fixing process of slide.
• After heat fix, whitish layer will be formed on slide.
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• After that we will put a few drop of primary

stain(crystal violet) on the slide and keep it for some
time.
• Now open the tap in very slow flow that slowly slowly
drop of water will remove excess colour.
• Again, Gram iodine will be put on the slide and again
wash that with slowly tap water.
• Now after that, Alcohol will be put on the slide.
• At the end we will put 4-6 drop of Saffranine and keep
it for 40-60 second. After that,put that slide on the
slow running tap for washing the slide.
• Now clean that with cotton and dry that.
• Now we will put it inside the microscope.
• It is oil immersion, so that we will put oil inside it to
take clear views.
▪ Results:
Gram-negative bacteria will stain pink/red and
Gram-positive bacteria will stain blue/purple.

▪ Discussion :
• After performing this, we could differentiate the
Gram positive and Gram negative Bacteria. Either
in the mean of shape or size of the Bacteria or in any
type. We can know the disease and we can test any
patient by this method.
• Peptidoglycan layer is rich in broken layer that is, It
consist of thicker Gram positive and Thinner Gram
negative Bacteria
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• Lipopolysaccharide is rich in Lipid layer which is

consist of thinner Gram positive and thicker Gram
negative Bacteria.