Gram staining is a method of
differentiating bacterial species into two large
groups(Gram-positive and Gram-negative).
The Gram staining is almost always the first step in the
identification of bacteria.
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 Gramindeterminate groups as well.
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.
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.
Cells will be
cells will retain
All Bacteria will be stained Purple
(Alcohol or Acetone)
Stain will be fixed due to formation of
complex of Crystal Violet & Iodine
Staining with Safranin(Counter Stain)
Cells that retains the colour of Primary Stain are Gram positive.
Cells that do not retains the colour of Primary Stain and takes up the colour of
Counter Stain are Gram Negative.
Applying a primary stain (Crystal Violet) to a heat-fixed
smear 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
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
Rinse the slide in running water and add
Again rinse the slide and cover the smear with the
Safranin for 1 min.
Wash off the safranin with water, air dry the slide and
Observe under oil immersion lens.