Gram stain is technique used to differntiate gram positive and gram negative bacteria. If you like this slideshare then please do share and follow me. You can visit my blogs:pranav2705.blogspot.com

1. GRAM STAINING.
PREPARED BY:
PRANAV PATEL.
PHARM D, SECOND YEAR.
KBIPER,GANDHINAGAR.

2. INTRODUCTION
• Gram stain or gram staining also called gram's
method is method of staining used to distinguish
and classify bacterial species into large two
groups:Gram positive and Gram negative bacteria.
• The name comes from the danish
bacteriologist Hans christian gram ,who
developed this technique

3. OBJECTIVE
• Objective of gram staining is to differntiate the bacteria into two groups:Gram positive
and Gram negative,which helps in classification and differntiation of microorganisms
• The gram stain separates bacteria into two groups:
1. Gram positive organisms which retain primary dye(Crsytal violet)
2. Gram negative organisms which take the colour of counterstain(Safranin)

4. PRINCIPLE
• Gram staining technique is based on differntial strucutre
of cellular membrane and cell wall of the organisms.
• Gram positive bacteria contains peptidoglycan thick cell
wall,but they do not have an outer membrane.While
gram negative bacteria contains peptidoglycan thin wall
with an outer

5. GRAM POSITIVE BACTERIA
• Peptidiglycan layer of gram positive bacteria retains the
primary dye,crystal violet(CV),with mordant iodine.Iodine and
crystal violet makes a complex within the
peptidoglycan.when decolorizer applied to cells CV-I complex
remains with cell and it shows dark purple to blue colour.
• Gram-positive bacteria take up the crystal violet stain used in
the test, and then appear to be purple-coloured when seen
through an optical microscope. This is because the
thick peptidoglycan layer in the bacterial cell wall retains
the stain after it is washed away from the rest of the sample,
in the decolorization stage of the test.

6. GRAM NEGATIVE BACTERIA
• Gram negative bacteria does not have thick
peptidoglycan layer.Following the application of crystal
violet and iodine,the CV-I complex is not trapped
within peptidoglycan.When acid-alcohol is decolorizer
dehydrates outer cellular membrane leaving holes in
the membrane and effectively washing CV-I complex
from cell.The cells appear colourless,so to make it
visible a secondary stain safranin is applied leaving
gram negative cells pink.

7. REAGENTS USED IN GRAM STAINNG
1. Primary stain:Crystal violet,95% ethyl alcohol,Ammonium oxalate,Distilled water.
2. Gram's iodine:Potassium iodide,Iodine crystals,Distilled water.
3. Decolorizer:Acetone and ethanol.
4. Counterstain:Safranin,Ethanol,Distilled water.

8. PROCEDURE
 Prepare and fix the specimen to the microscope slide before staining.
 Cover the smear with crystal violet, the primary stain, for 20 seconds.
 Gently rinse off the stain with water.
 Cover the smear with Gram’s iodine, the mordant, for 1 minute.
 Pour off the excess Gram’s iodine.
 Run the acid-alcohol decolorizer over the smear until the solution appears clear.
 Gently rinse with water.
 Cover the smear with safranin, the secondary or counterstain, for 20 seconds.
 Gently rinse the stain with water.

9. RESULT
• Gram Positive bacteria:Blue/Purple colour.
• Gram Negative bacteria:Red/Pink colour.

10. DIFFERENCE BETWEEN GRAM POSITIVE AND GRAM
NEGATIVE BACTERIA
• As Gram positive bacteria lack an outer lipid
membrane, when correctly referring to their
structure rather than staining properties, are
termed monoderms. The outer lipid membrane
possessed by Gram negative bacteria means that,
when referring to their physical structure, they are
termed diderms.

11. KEY CHARACTERS DIFFERNCE
S.N. Character Gram positive bacteria Gram negative bacteria.
1 Cell wall thickness Thick(20-80nm) Thin(8-10nm)
2 Peptidoglycan layer Thick Thin
3 Outer membrane Absent Present
4 Porins Absent Present
5 Ratio of RNA:DNA 8:1 Almost 1
6 Pathogens Few pathogenic bacteria
belong to gram positive
group
Most pathogens are gram
negative
7 Examples Staphylococcus
Streptococcus
Bacillus
Clostridum
Escherichia
Salmonella
Proteus
Helicobacter

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