GROUP 1(ONE)

GRAM STAIN
 Group Members( College ID)
❖ Idrissa Conteh (9271) – Group Lead
❖ Solomon Fallah Foa SANDY (9375)
❖ Mamusu Halimatu Karim Mattia (9239)
❖ Francylda Cummings-John (9164)
❖ Musa V. Bona (9407)
❖ Hassan Samu (9373)
❖ Osman Ibrahim Kamara (9224)
❖ Constance U.M. M’bayo (9227)
❖ Rugiatu Karima Kamara (9273)
❖ Mariama Mansaray (9440)
❖ Victoria Wisman (9212)
 Presentation Outline
❖Introduction
❖Brief History
❖Procedure
❖Interpretation
❖Principle
❖Modifications
❖Uses
❖Limitations
 Introduction
➢BACTERIAL STAINING: Is the process of coloring of
colorless bacterial structural components using stains
(dyes).
➢GRAM STAINING is a differential staining technique used to
classify bacteria. By the use of Gram Stains, Bacteria are
classified into either
❑Gram Negative
❑Gram Positive
based on the chemical and physical properties of their cell walls
Brief History
➢This staining technique was originally developed
by Hans Christian Gram (1884) while studying
lung tissue sections from a patient who died of
pneumonia.
➢Carl Weigert (1845 – 1904) added the final step
of staining with safranin
➢Even after more than 130 years of its discovery
and even if the newer modern diagnostic facilities
are available, still Gram stain remains most
widely used stain in diagnostic bacteriology.
 Procedure
❖Gram Staining is done in the
following steps

➢ Fixation
➢ Primary stain
➢ Mordant
➢ Decolorization
➢ Counter Stain
 Fixation
❖Fixation: The preservation and hardening of a tissue
sample to retain as nearly as possible the same relations
they had in the living body

➢The smear made on a

slide from bacterial
culture or specimen, is
air dried and then heat
fixed.
 Primary Stain
➢ The smear is stained with
Pararosaniline dyes such as
crystal violet for one minute.
Then the slide is rinsed with
water.

Crystal violet stains all the bacteria

violet in color
Mordant
❖ MORDANT: Is a substance which when taken up by the
microbial cells help make dye in return, serving as a link or
bridge to make the staining process possible e.g. iodine.

➢Gram's iodine is poured over

the slide for one minute.
➢Then the slide is rinsed with
water.

Gram's iodine acts as a mordant.

 Decolorization
➢Next step is pouring of few drops of decolorizer to the smear:
➢Slide is immediately rinsed with water.
➢Decolorizer removes the primary stain from gram negative
bacteria while the gram-positive bacteria retain the primary
stain.
Decolorization cont………….
Note: Decolorization is the most crucial step of Gram
stain. If the decolorizer is poured for more time, even
gram positive bacteria loose color and if poured for
less time, the gram negative bacteria do not lose the
color of primary stain properly

Examples of Decolorizer includes:

o Acetone (for 1-2 sec)
o Ethanol (20-30 sec)
o Acetone alcohol (for 10 sec)
o Iodine acetone.
 Counter stain
• Secondary stains such as safranin or diluted
carbol fuchsin is added for 30 seconds. it
• Imparts pink or red color to the gram-
negative bacteria.
• Alternatively, neutral red may also be used
as counter stain, especially for gonococci.
• The slide is rinsed in tap water, dried, and
then examined under oil immersion
objective.
Interpretation
Smear is examined under oil
immersion objective.
➢ Gram-positive resist decolorization
and retain the color of primary stain
i.e. violet.
➢Gram-negative bacteria are
decolorized and, therefore, take
counter stain and appear pink.
Principle of Gram Stain
➢Though the exact mechanism is not understood,
the Following theories have been put forward
➢ pH theory: Cytoplasm of gram-positive bacteria is
more acidic, hence, can retain the basic dye (e.g.
crystal violet) for longer time.
➢ Gram Iodine: serves as mordant, i.e. it combines
with the primary stain to form a dye-iodine
complex which gets retained inside the cell.
 Principle of Gram Stain Cont…….
Cell wall theory:
This is believed to be the most important postulate
to describe the mechanism of Gram stain.
❑ Gram-positive cell wall has a thick peptidoglycan
layer (50-100 layers thick), which are tightly cross
linked to each other.
❑ The peptidoglycan itself is not stained; instead, it
seems to act as a permeability barrier preventing
loss of crystal violet. More so, alcohol is thought
to shrink the pores of the thick peptidoglycan.
Principle of Gram Stain Cont…….
❑Gram-negative cell wall is more
permeable thus allowing die out flow of
crystal violet easily. this is attributed to:-
❑The thin peptidoglycan layer in gram-
negative cell wall which is not tightly
cross linked. Presence of
lipopolysaccharide layer in the cell wall of
gram-negative bacteria, which gets
disrupted easily by die action of acetone
or alcohol; thus allowing die primary stain
to come out of the cytoplasm.
 Principle of Gram Stain Cont…….
• After mordanting with Gram's iodine, bigger dye-
iodine complexes are formed in the cytoplasm.
• Following decolorization, as more lipid content in
gram-negative bacterial cell wall gets dissolved
leading to formation of larger pores through which
the dye-iodine complexes escape.
• Due to less lipid in gram-positive bacterial cell wall
smaller pores are formed which do not allow the
dye- iodine complexes 10 escape .

✓ Cell Wall
✓ Outer Membrane
✓ Periplasmic space
✓ Flagella structure
✓ Toxins produced
✓ Resistance to physical
disruption
✓ Inhibition by basic dyes
✓ Mesosome
✓ Antibiotic Resistance
✓ Susceptibility to anionic
detergents
✓ Resistance to sodium
azide
GRAM-NEGATIVE BACTERIA GRAM-POSITIVE BACTERIA
Has a thin peptidoglycan layer (single-layered) Thick peptidoglycan layer(multilayered)
High lipopolysaccharide Teichoic Acid
Phospholipid
Lipoprotein (high)
Present Absent
Present (more prominent) Present(less prominent)
4 rings in basal body 2 rings in basal body
Primarily endotoxins Primarily exotoxins
low High
low High
Less prominent More prominent
More resistant to antibiotics More susceptible to antibiotics
low High
low High
Modifications of Gram Stain
❖There are few minor modifications of Gram stain which
vary slightly from the method described earlier.
❑Kopeloff and Beerman's modification: Primary stain and
counter stain used are methyl violet and basic fuchsin
respectively.
❑Jensen's modification: This method involves use of
absolute alcohol as decolorizer and neutral red as counter
stain. lt is useful for meningococci and gonococci.
 Modifications of Gram Stain Cont………
❑Weigert's modification: This modification is useful
for staining tissue sections. Here, aniline-xylol is
used as a decolorizer.
❑Preston and Morrell's modification: Here, iodine-
acetone is used as decolorizer.
 Uses of Gram Stain
✓ Differentiation of bacteria into gram-positive and Gram-negative:
✓ For identification: Gram staining from bacterial culture gives an idea to put
the corresponding biochemical test for further identification of bacteria
✓ To start empirical treatment: Gram stain from specimen gives a preliminary
clue about the bacteria present (based on the shape and Gram staining
property of the bacteria) so that the empirical treatment with broad
spectrum antibiotics can be started early before the culture report is
available.
✓ It influences the choice of antibiotic because, in general, gram-positive
bacteria are more susceptible to penicillin G than are gram-negative bacteria
Uses of Gram Stain Cont…
➢For fastidious organisms, such as Haemophilus which
takes time to grow in culture; Gram stain helps in early
presumptive identification .
➢Anaerobic organisms, such as Clostridium, which do not
grow in routine culture. Hence, Gram stain gives a
preliminary clue to put anaerobic culture.
➢Yeasts: In addition to stain the bacteria, Gram stain is
useful for staining certain fungi such as Candida and
Cryptococcus (appear gram-positive).
Limitations of Gram Stain
❑Some Gram-positive bacteria may lose the stain easily and therefore
appear as a mixture of Gram-positive and Gram-negative bacteria (gram
variable)
❑Old cultures of Gram positive bacteria where cell walls may be
weakened may readily get decolorized.
❑Gram-positive cells affected by cell wall active agents such as lysozyme
or antibiotics may become gram negative.
❑Gram positive bacteria that have been phagocytized by polymorphs may
also appear Gram negative
❑Bacteria devoid of cell wall are always gram negative e.g. mycoplasma
Note
❑However, not all bacteria can be seen in the Gram stain.
Medically important bacteria that cannot be seen and describes
the reason why. The alternative microscopic approach to the
Gram stain is also described.
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