2. 2
Gram Stain:Gram Stain:
It is the most important
differential stain used in
bacteriology classifies
bacteria into two major
groups::
a)a)Gram positive:Gram positive:
Appears violet after
Gram’s stain
b)b) Gram negative:Gram negative:
Appears pink after Gram’s
stain
3. Developed by Hans Christian Gram (1884)
Differential staining technique two stains
are used to impart different colours to different
bacteria
Other differential stains:
Acid-fast Stain
Albert Stain
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4. PROCEDURE OF GRAM STAIN
1. Fixation: smear made on slide from bacterial culture air
dried heat fixed
2. Primary stain: stained with crystal violet. Slide rinsed with
water. All bacteria stained violet
3. Mordant: gram’s iodine poured over slide and kept for 1
min. Slide rinsed with water.
4. Decolourization: few drops of decolourizer to smear i.e.
ethanol (20-30 sec). Slide rinsed with water.
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5. • Removes the primary stain from gram-negative bacteria. Most
important step
5. Counter stain: secondary stains safranin added for 30 sec.
Gives pink/red colour to gram-negative bacteria.
Slide rinsed with water, dried and examined under oil
immersion objective
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11. PRINCIPLE OF GRAM STAIN
• Based upon two theories:
A. Cell Wall Theory
Gram-positive bacteria
• Have a thick peptidoglycan layer surrounds the cell.
• The stain gets trapped into this layer and the bacteria turned
violet
• Retain the color of the primary stain (crystal violet) after
decolorization with alcohol
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12. Gram-negative bacteria
• have a thin peptidoglycan layer that does not retain crystal
violet stain.
• Instead, it has a thick lipid layer which dissolved easily upon
decolorization with Acetone/Alcohol allow primary stain to
come out of cytoplasm
• Therefore, cells will be counterstained with safranin and turned
pink.
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14. B. pH Theory
• Cytoplasm of gram-negative bacteria is more acidic
and can therefore retain the basic dye i.e. crystal
violet.
• Iodine acts mordant and form dye-iodine complex
retained in cell
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15. Disadvantages of Gram-Stain
Some bacteria are Gram stain variable
(positive or negative results)
Some bacteria are resistant to Gram
stain (i.e. acid-fast bacteria)
False results may occur if over-
decolorized
Older cultures may give false results15
16. MODIFICATIONS OF GRAM-STAIN
Kopeloff and Beerman’s modification
Primary stain methyl red and counterstain basic
fuschin
Jensen’s Modification
Absolute alcohol used as declourizer and neutral red as
counterstain
Used for meningococci and gonnococci
Weigert’s Modification
Aniline-xylol used as decolourizer. Used for staining
tissue sections
Preston and Morrell’s Modification
Iodine-acetone used as decolourizer 16
17. APPLICATION/ USES OF GRAM-STAIN
1. Differentiation of bacteria into gram-positive and
gram-negative first step of identification
2. Helps with rapid diagnosis in critical conditions such
as meningitis
3. Choice of medicine gram-stain gives possible clue
of what the organism might be (gram-negative/gram-
positive). Doctor can start treament with broad
spectrum antiobiotic
4. Fastidious take time to grow organisms but gram-
stain help in presumptive identification
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18. 5. Anaerobic organisms do not grow in routine culture, gives
clue to put for anerobic culture e.g. Clostriduim
6. Yeasts gram-stain useful for staining certain fungi e.g.
Candida and Cryptococcus
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