Gram stain

It is one of the most important staining

..techniques in microbiology

It is almost always the first test performed for

..the identification of bacteria

Gram developed a staining procedure which

divided almost all bacteria into two large
groups ..(Gram-positive and Gram-negative)
 Gram positive and negative
We first must to know what the
.. Different between

Gram positive and

…gram negative cells
 Gram positive cell
The gram-positive cell envelope consists of
a thick layer of peptidoglycan embedded
with techoic acids and a plasma
membrane comprised of phospholipids
with integral membrane proteins
..traversing the bilayer
 Gram negative cell
The gram-negative cell envelope consists of
a thin layer of peptidoglycan surrounded
by two phospholipid membranes, one
.interior and one exterior

Polysaccharide chains are bound to the

phosphate heads of the outer membrane
..to form lipopolysaccharides
Both the membranes
contain integral
membrane
.proteins
Place cursor over
each membrane
…for ID
Gram staining is based on the ability of
bacteria cell wall to retaining the crystal
..violet dye during solvent treatment
 Gram stain procedure
Place a slide with a bacterial smear on a**
staining rack
stain the slide with**
crystal violet
.for 30-60 sec
Pour off the stain**

Flood slide with**

Gram's iodine
for 1-2 min

..Pour off the iodine**

 Decolourize by washing the slide**
..briefly with acetone (2-3 seconds)

Wash slide**
thoroughly with
water to remove
– the acetone
do not delay
..with this step
 Flood slide with**
safranin
counterstain
..for 30 sec

..Wash with water**

Blot excess water**
and dry in hand
..over bunsen flame
What’s happen to gram negative cell
.. when we stain it
The cells are flooded**
.with crystal violet dye
The individual crystal***
violet ions penetrate the thin peptidoglycan
layer of the cell as well as the plasma
membrane, making their way through the
matrix created by the crosslinking of
polysaccharides and proteins within the
..peptidoglycan layer
Gram's iodine is added and penetrate the **
thin peptidoglycan layer of the cell. the
..iodide ions mix with the crystal violet dye

The crystal violet and iodide ions react,**

forming a crystal violet-iodine complex.
This complex is insoluble in water and
produces particles much larger than either
the iodide ions or the crystal violet ions
.individually
A decolorizing solution, normally consisting**
of a mixture of ethyl alcohol and acetone,
.is added
The mixture displaces water in the**
peptidoglycan layer, resulting in
dehydration. This loss of water causes the
thin peptidoglycan layer to shrink slightly,
tightening the matrix created by the
crosslinking of polysaccharides and
proteins. The mixture also disrupts and
dissolves the outer membrane, exposing
the peptidoglycan layer to the
.environment
Although the thin peptidoglycan layer of the
gram-negative envelope is dehydrated,
the crystal violet-iodide complex can
escape through the large pores that
remain. The complex is eventually washed
away, leaving colorless, unstained cells,
unlike gram-positive cells which appear
.purple at this step
The counterstain, normally safranin, is **
.added
 What’s happen to gram positive cell
.. when we stain it
The cells are flooded with crystal violet**
dye. Crystal violet is a water-soluble, basic
dye
The individual crystal violet ions penetrate **
the thick peptidoglycan layer of the cell as
.well as the plasma membrane
Gram's iodine solution is added**
the iodide ions are also able to penetrate **
the thick peptidoglycan layer of the cell
The crystal violet and iodide ions forming
.. large complex
 .decolorizing solution, is added
It displaces water in the peptidoglycan
layer, resulting in dehydration. This loss of
water causes the thick peptidoglycan layer
to shrink, tightening the matrix created by
the crosslinking of polysaccharides and
.proteins
Because of its larger size, the crystal violet-
iodine complex is blocked from moving
easily through the thick layers of
dehydrated peptidoglycan and exiting the
.bacterial cell
The counterstain, normally safranin, is**
added
because of its small size
of safranin is able
to penetrate the
dehydrated
peptidoglycan layer

When viewed under a microscope, gram-**

positive cells appear purple due to the
crystal violet-iodine complex retained
inside
The end