Gram stain methods

Made by : DR ( Rasha Bennour)

Supervised by : Associate professor ( Omra O. Bugrein,
MD )
Overview :
• The most widely used staining
procedure in microbiology is the
Gram stain, discovered by the
Danish scientist and physician Hans
Christian Joachim Gram in 1884.to
differentiate Pneumococci from
Klebsiella Pneumonia. It is also
known as “ Differential staining”, as it
divides micro-organisms into two
maingroups i.e. Gram’s +ve and
Hans Christian Joachim Gram
 Importance of a Gram Stain:
• The Gram stain is a very important step in the
initial characterization and classification of
bacteria. It is also a key procedure in the
identification of bacteria based on staining
characteristics, enabling the bacteria to be
examined using a light microscope. The
bacteria present in an unstained smear are
invisible when viewed using a light
microscope. Once stained, the morphology
and arrangement of the bacteria may be
observed as well. Furthermore, it is also an
important step in the screening of infectious
•This differential staining procedure separates
most bacteria into two groups on the basis of cell
wall composition:
1- Gram positive bacteria (thick layer of
peptidoglycan-90% of cell wall)- stains purple.
2- Gram negative bacteria (thin layer of
peptidoglycan-10% of cell wall and high lipid
content) –stains red/pink.
 PRINCIPLE OF GRAM STAINING
• The structure of the organism’s cell
wall determines whether the organism is gram
positive or negative. When stained with a
primary stain and fixed by a mordant, some
bacteria are able to retain the primary stain by
resisting declorization while others get
decolorized by a decolorizer. Those bacteria
which retain the primary stain are called Gram
positive and those bacteria which gets
decolorized and then get counterstained are
called Gram negative.
 REQUIREMENTS AND PREPARATION
OF REAGENTS:
• 1-Primary Stain : Crystal violet
Solution A :
• 1_Crystal violet = 2 gm
• 2_Ethyl alcohol= 20 ml
• Solution B :
• 1_Ammonium oxalate = 0.8 gm
• 2_Distilled water = 80 ml
• Mix solution A and B. Keep for 24 hours and
filter. Store in an amber colored bottle.
• 2-Mordant : Gram’s Iodine
– Iodine = 1 gm
– Potassium iodide = 2 gm
– Distilled water = to 100 ml
• Mix and Store in an amber colored bottle.

• 3-Decolorizer : 95% Ethanol or 1:1 acetone with ethanol

– Acetone = 50 ml
– Ethanol (95%) = 50ml
• 4-Counterstain: safranin
– Safranin O = 0.34 gm
– Absolute alcohol = 10ml
– Distilled water = 90ml

• Mix, filter and store in ambered

• colored bottle.
Crystal violet (CV) dissociates into CV+ and Cl– ions in
aqueous solutions. These ions penetrate through the cell
wall and cell membrane of both Gram-positive and
Gram-negative cells. The CV+ ion interacts with
negatively charged components of bacterial cells and
stains the cells purple.

Iodine (I), used as mordant interacts with CV+ and

forms large complexes of crystal violet and iodine (CV–I)
within the inner and outer layers of the cell.

When a decolorizer such as alcohol or acetone is

added, it interacts with the lipids of the cell membrane.
Since Gram negative organism have thin
peptidoglycan layer(1-2 layers) and have
additional lipopolysaccharide layer which gets
 so gram negative organism fails to retain the complex
and gets decolorized as the complex is washed away
In contrast, a Gram-positive cell becomes dehydrated
from an ethanol treatment. This closes the pores in the
cell wall and prevents the stain from exiting the cell. The
large CV–I complexes become trapped within the Gram-
positive cell also due to the thick and multilayered (40
layers) nature of its peptidoglycan.

After decolorization, the Gram-positive cell remains

purple and the Gram-negative cell loses its purple color.
Counterstain, which is usually positively-
charged safranin or basic fuchsin, is applied last to
give decolorized Gram-negative bacteria a pink or red
color.
 PROCEDURE OF GRAM STAINING
• Smear preparation :
1-Take a grease free dry slide.
2-Sterilize the inoculating loop on a flame.
3-Transfer a loopful of culture (or the
specimen) by sterile loop and make a
smear at the center. Smear should not be
very thin or very thick.
4-Allow the smeat to dry in the air.
5-Fix the dry smear by passing the slide 3-4
times through the flame quickly with the
smear side facing up.
Gram Staining :
1-Place the slides on the staining rods.
2-Cover the smear with crystal violet stain
and leave for 1 minute.
3-Wash carefully under running tap water.
4-Flood the smear with Gram’s iodine solution
and leave for 1 minute.

5-Drain off the iodine Wash the slide for the

again in a gentle stream of tap water.

6-Flood the slide with the decolorizing agent

then wait for 20-30 seconds. This can also
be done by adding a drop by drop to the
slide until the decolorizing agent running
from the slides runs clear.
.

7-Gently wash the slide under running tap

water and drain completely.

8-Counterstain with safranin for and and

wait for about 30 seconds to 1 minute.

9-Wash slide in a gentile and indirect

stream of tap water until no color appears
in the effluent and then blot dry with
absorbent paper.
Bacterial Morphology:
 MCQs
• 1. What is the correct order of staining
reagents in Gram-Staining?
a) Crystal violet, alcohol, iodine solution,
safranin.
b) Crystal violet, iodine solution, alcohol,
safranin.
c) Crystal violet, safranin, alcohol, iodine
solution.
d) Iodine solution, crystal violet, alcohol,
safranin.
Answer: b

Explanation: Gram staining is a

type of differential staining. In this
process the fixed bacterial smear is
subjected to the following staining
reagents in the order listed: crystal
violet, iodine solution, alcohol
(decolorizing agent), and safranin.
• 2-Which of the following are true for Gram-
negative bacteria?

a) upon alcohol treatment, the permeability of

the cell wall increases.

b) crystal violet-iodine (CV-I) complex is

extracted.

c) pore size decreases and the CV-I complex

cannot be extracted.

d) alcohol treatment increases the permeability

of the cell wall and the CV-I complex can be
extracted.
• Answer: d

Explanation: Experimental evidence

suggests that during staining of Gram-
negative bacteria the alcohol treatment
extracts the lipid, which results in
increased permeability of the cell wall.
Thus the crystal violet-iodine (CV-I)
complex can be extracted and the Gram-
negative organism is decolorized. These
cells subsequently take on the color of the
safranin counterstain.
• 3. In Gram-staining, iodine is used as
a______________

a) fixative
b) mordant
c) solublizer
d) stain
• Answer: b

Explanation: In Gram-staining, iodine acts

as a mordant i.e. it combines with the dye
or stain and thereby fixes it on the
material. It increases the interaction
between stain solution and the bacterial
cell.
THANK YOU FOR YOU
ATTETION