The document provides information about the Gram stain procedure and its importance in bacteriology. It describes how the Gram stain technique classifies bacteria into two groups - Gram positive and Gram negative - based on whether they appear violet or pink after staining. Key steps include fixing a smear, staining with crystal violet and iodine, decolorizing with alcohol or acetone, and counterstaining with safranin. The principle is that Gram positive bacteria have a thick peptidoglycan layer that retains the primary stain, while Gram negative bacteria have a thin layer that is decolorized, taking up the counterstain instead. The Gram stain is a critical first step in bacterial identification and diagnosis.

1. GRAM-STAIN PRACTICAL BRIEFING
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Ms. Clemencia Tjazuko
MSc Medical Microbiology

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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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Step 1:Step 1: Crystal Violet
Step 2:Step 2: Gram’s IodineGram’s Iodine
Step 3:Step 3: DecolorizationDecolorization
(Aceton-Alcohol)(Aceton-Alcohol)
Step 4:Step 4: Safranin RedSafranin Red

9. RESULTS
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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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