The Gram staining method is used to differentiate between Gram-positive and Gram-negative bacteria. It was developed in 1884 by Hans Christian Gram who discovered that certain stains were preferentially retained by bacterial cells. The method involves applying a primary stain, adding a mordant to trap the stain, decolorizing with alcohol or acetone, and counterstaining - bacteria that retain the primary stain are Gram-positive while those that take up the counterstain are Gram-negative. Though a valuable diagnostic tool, not all bacteria can be definitively classified by Gram staining.

1. GRAM STAINING METHOD
FOR BACTERIA

2. Gram staining is a method of differentiating
bacterial species into two large groups (Gram-
positive and Gram-negative).
It is a valuable diagnostic tool in both clinical and
research settings, not all bacteria can be definitively
classified by this technique.
This gives rise to Gram-variable and Gram
indeterminate groups as well.

3. The method is named after its inventor, the Danish
scientist Hans Christian Gram (1853–1938), who
developed the technique while working with Carl
Friedländer in the morgue of the city hospital in Berlin
in 1884.
In 1884, while examining lung tissue from patients who
had died of pneumonia, Gram had discovered that
certain stains were preferentially taken up and retained
by bacterial cells.

4. Gram did not use a counterstain in his procedure.
It was a few years later, that the German
pathologist Carl Weigert(1845-1904) from
Frankfurt, added a final step of staining with
Safranin.
Gram himself never used the red counterstaining
in order to visualize the gram negative bacteria

6. Methylene blue or basic fuchsin are used
They provide colour contrast but impart the
same colour to all bacteria

7. Indian ink or nigrosin are used.
They produce uniformly coloured background against
which the unstained bacteria stand out in contrast.
Particularly useful in the demonstration of bacterial
capsule which do not take simple stain and also for
spirochetes.

8. These stains impart different colours to different
bacteria or bacterial structures
Primary staining is done by one dye
Counter staining is done by a different dye of
contrasting colour
Example- Gram stain and Ziehl-Neelsen stain

9.  Silver impregnation method
 Used for structures and cells too thin to be seen under
the ordinary microscope like spirochetes and bacterial
flagella
 They may be rendered visible if they are thickened by
impregnation of silver on their surface
 Example- Fontana’s and Levaditi’s methods of staining

10. Bacteria All Bacteria will be stained Purple
Cells will be
Decolourized Stain will be fixed due to formation of
complex of Crystal Violet & Iodine
Saffranin
Cells retain the color of primary stain are gram positive
Cells do not retain color of primary stain are gram positive but takes up the
color of counter stain are gram negative
Stained with
Crystal Violet
Gram Iodine
solution
Alcohol or
Acetone

13. Applying a primary stain (Crystal Violet) to a
heat-fixed mear of a bacterial culture.
The addition of Grams Iodine, which binds to
crystal violet and traps it in the cell.
Decolourization with Alcohol or Acetone, and
Counter staining with Safranin

14.  Prepare a heat fixed smear of the bacterial culture
 Cover the smear with the Crystal Violet for 1 min.
 Add Grams Iodine, which washes the crystal violet stain
 Rinse the slide in running water and add
decolourizer(Alcohol)
 Again rinse the slide and cover the smear with the
Safranin for 1 min.
 Wash off the safranin with water, air dry the slide
 Observe under oil immersion lens

15. We know that Gram positivity is restricted almost
exclusively to the bacteria, with only a few other
groups, such as the yeasts, exhibiting this reaction.

16. THANK YOU
FOR
ATTENTION