Gram staining is a method used to classify bacterial species into two groups: gram-positive and gram-negative. It involves staining a bacterial smear with crystal violet dye, iodine, decolorizer, and safranin counterstain. Gram-positive bacteria appear purple after staining due to the thickness of their cell walls retaining the crystal violet dye. Gram-negative bacteria appear pink since their thinner cell walls are decolorized by the alcohol or acetone wash, leaving only the safranin counterstain visible. While Gram staining is a standard first step in bacterial identification, not all bacteria can be definitively classified by this technique alone due to variations in cell wall composition.

1. {
GRAM’S STANNING:
Presented by:
Khushbu’s Microbiology

2. Purpose of Gram Staining
Gram stain or Gram staining, also
called Gram's method, is a method
of staining used to distinguish and
classify bacterial species into two large
groups: gram-positive
bacteria and gram-negative bacteria.

3. Danish bacterio
logist Hans Christian Gram who discovered
Gram Stating method.
Gram stain, still a standard technique to classify
the bacteria.

4.  Prepared thin smear of microorganism and dry in
air and fix with gentle heat.
 Flood with Crystal violet for 01 minute.
 Wash with water and flood with gram’s Iodine for 01
minute.
 Wash with the water and decolorize with gram’s
decolorize until no further violet color comes off.
 Wash with water and counter stain with 0.5 %
safranin for 1 minute.
 Wash with water, dry it.
 Observe under oil immersion objective in
Microscope
Methodology of Gram’s
staining

6. Gram staining differentiates bacteria by the
chemical and physical properties of their cell
walls.
Gram staining is almost always the first step in
the preliminary identification of a bacterial
organism.
While Gram staining is a valuable diagnostic tool
in both clinical and research settings, not all
bacteria can be definitively classified by this
technique.
Application of
Gram’s Stating

7. Application of Reagent
Cell color
Gram-positive Gram-negative
Primary dye crystal violet purple purple
Trapping agent iodine purple purple
Decolorizer alcohol/acetone purple colorless
Counter stain
safranin/carbol
fuchsin
purple pink
Summary of Gram stain
Summary of Gram stain

8. THANKS