Gram Staining Principle, Procedure, Int

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Gram Staining :
Principle, Procedure,
Interpretation and
Animation
Posted on January 19, 2016 by Dhurba Giri in
Bacteriology, Microbiology // 0 Comments
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The Gram staining technique is the most

important and widely used
microbiological dierential staining
technique. It was developed by Dr.
Christian Gram in 1884, and categorizes
bacteria according to their Gram character
(Gram positive or Gram negative). In
addition this stain also allows
determination of cell morphology, size,
and arrangement. It is typically the rst
dierential test run on a specimen
brought into the laboratory for
identication. In some cases, a rapid,
presumptive identication of the
organism or elimination of a particular
organism is possible.

PRINCIPLE OF GRAM STAINING

The structure of the organisms cell wall
determines whether the organism is gram
psitive or negative. When stained with a
primary stain and xed 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.

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 (CVI) 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 dissolved due to the addition
of alcohol, 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 CVI 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.

REQUIREMENTS AND
PREPARATION OF REAGENTS
1. Primary Stain : Crystal violet

Solution A :

Crystal violet = 2 gm
Ethyl alcohol= 20 ml

Solution B :

Ammonium oxalate =
0.8 gm
Distilled water = 80 ml

Mix solution A and B. Keep for

24 hours and lter. Store in an
amber colored bottle.
 2. Mordant : Grams 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, lter and store in ambered

colored bottle.

PROCEDURE OF GRAM
STAINING
Smear preparation :
1. Take a grease free dry slide.
2. Sterilize the inoculating loop on a
ame of a Bunsen burner.
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 ame
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 Grams iodine
solution and leave for 1 minute.
5. Drain o 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 euent and then
blot dry with absorbent paper.
10. Observe under microscope.
INTERPRETATION OF GRAM
STAINING

The staining results of gram stain are as

follows :

Gram Positive : Dark purple

Gram Negative : Pale to dark red
Yeasts : Dark purple
Epithelial cells : Pale red

Examples of Gram Positive

Organisms
Bacillus, Nocardia, Clostridium,
Propionibacterium, Actinomyces,
Enterococcus, Cornyebacterium, Listria,
Lactobacillus, Gardnerella, Mycoplasma,
Staphylococcus, Streptomyces, Streptococcus
etc
Examples of Gram Positive
Organisms
Escherichia, Helicobcater, Hemophilus,
Neisseria, Klebsiella, Enterobacter,
Chlamydia, Vibrio, Pseudomonas,
Salmonella, Shigella

ANIMATION OF GRAM
STAINING

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About Dhurba
Giri
Dhurba Giri is the
author at
LaboratoryInfo.com,
a scientic blog dedicated for Medical
Laboratory Professionals. He's
currently studying Bachelor in Medical
Laboratory Technology nal year at
Pokhara University, Nepal. Connect
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