Transcript Gram stain

‫‪Gram stain‬‬
‫عمل الطالبة ‪ :‬ديانا شكري القدرة‬
‫بإشراف الدكتور‪ :‬عبد الرؤوف‬
History of the Gram
In 1884, Hans Christian Gram, a Danish
doctor working in Berlin, accidentally
stumbled on a method which still forms
the basis for the identification of bacteria.
While examining lung tissue from patients
who had died of pneumonia
The Gram staining method, named after the
Danish bacteriologist who originally
devised it
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
)Gram-negative and Gram-positive(groups ..
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 violetiodine 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, grampositive cells appear purple due to the
crystal violet-iodine complex retained
inside
The end