Date:- 04-06-2021

Practical:- Gram’s staining

Introduction
The Gram stain is the most widely used staining procedure in bacteriology. It is called a
differential stain since it differentiates between Gram-positive and Gram-negative bacteria.
Bacteria that stain purple with the Gram-staining procedure are termed Gram-positive;
those that stain pink are said to be Gramnegative. The terms positive and negative have
nothing to do with electrical charge, but simply designate 2 distinct morphological groups of
bacteria.
Gram-positive and Gram-negative bacteria stain differently because of fundamental
differences in the structure of their cell walls. The bacterial cell wall serves to give the
organism its size and shape, as well as to prevent osmotic lysis. The material in the bacterial
cell wall that confers rigidity is peptidoglycan.
In electron micrographs, the Gram-positive cell wall appears as a broad, dense wall 20–80
nm thick and consists of numerous interconnecting layers of peptidoglycan. Chemically, 60%
to 90% of the Gram-positive cell wall is peptidoglycan. Interwoven in the cell wall of Gram-
positive are teichoic acids. Teichoic acids, that extend through and beyond the rest of the
cell wall, are composed of polymers of glycerol, phosphates, and the sugar alcohol ribitol.
Some have a lipid attached (lipoteichoic acid). The outer surface of the peptidoglycan is
studded with proteins that differ with the strain and species of the bacterium.
The Gram-negative cell wall, on the other hand, contains only 2–3 layers of peptidoglycan
and is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide,
lipoprotein, and proteins. Only 10%–20% of the Gram-negative cell wall is peptidoglycan.
The phospholipids are located mainly in the inner layer of the outer membrane, as are the
lipoproteins that connect the outer membrane to the peptidoglycan. The
lipopolysaccharides, located in the outer layer of the outer membrane, consist of a lipid
portion called lipid A embedded in the membrane, and a polysaccharide portion extending
outward from the bacterial surface. The outer membrane also contains a number of
proteins that differ with the strain and species of the bacterium.
The Gram-staining procedure involves 4 basic steps:
1. The bacteria are first stained with the basic dye crystal violet. Both Grampositive and
Gram-negative bacteria become directly stained and appear purple after this step.
2. The bacteria are then treated with Gram’s iodine solution. This allows the stain to be
retained better by forming an insoluble crystal violet-iodine complex. Both Gram-positive
and Gram-negative bacteria remain purple after this step.
3. Gram’s decolorizer, a mixture of ethyl alcohol and acetone, is then added. This is the
differential step. Gram-positive bacteria retain the crystal violet iodine complex, while
Gram-negative are decolorized.
4. Finally, the counter stain safranin (also a basic dye) is applied. Since the Gram-positive
bacteria are already stained purple, they are not affected by the counter stain. Gram-
negative bacteria, which are now colorless, become directly stained by the safranin. Thus,
Gram-positive bacteria appear purple and Gram-negative bacteria appear pink.

Principle
With the current theory behind Gram-staining, it is thought that in Grampositive bacteria,
the crystal violet and iodine combine to form a larger molecule that precipitates out within
the cell. The alcohol/acetone mixture then causes dehydration of the multilayered
peptidoglycan, thus decreasing the space between the molecules and causing the cell wall
to trap the crystal violet-iodine complex within the cell. In the case of Gram-negative
bacteria, the alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane
of the cell wall and may also damage the cytoplasmic membrane to which the peptidoglycan
is attached. The single thin layer of peptidoglycan is unable to retain the crystal violet iodine
complex and the cell is decolorized.

Requirements
crystal violet , Gram’s iodine solution, ethyl alcohol and acetone (mixture), safranin,
distilled water, bunsen burner, inoculation loop, Trypticase soy agar plate cultures of
Escherichia coli (a small, Gram-negative rod) and Staphylococcus epidermidis (a Gram-
positive coccus in irregular, often grapelike clusters).
Procedure
1. Heat-fix a smear of a mixture of Escherichia coli and Staphylococcus epidermidis
as follows:
(a) Using the dropper bottle of distilled water found in your staining rack, place a small
drop of water on a clean slide by touching the dropper to the slide.
(b) Aseptically remove a small amount of Staphylococcus epidermidis from the agar
surface and mix it generously with the water. Flame the loop and let it cool. Now,
aseptically remove a small amount of Escherichia coli and sparingly add it to the water.
Flame the loop and let it cool.
(c) Using the loop, spread the mixture over the entire slide to form a thin film.
(d) Allow this thin suspension to completely air dry.
(e) Pass the slide (film-side up) through the flame of the bunsen burner 3or 4 times to
heat-fix.
2. Stain with Hucker’s crystal violet for 1 minute. Gently wash with water. Shake off the
excess water, but do not blot dry between steps.
3. Stain with Gram’s iodine solution for 1 minute and gently wash with water.
4. Decolorize by adding Gram’s decolorizer drop by drop until the purple stops flowing.
Wash immediately with water.
5. Stain with safranin for 1 minute and wash with water.
6. Blot dry and observe using oil immersion microscopy.

Note
It is important to note that Gram-positivity (the ability to retain the purple crystal violet-
iodine complex) is not an all-or-nothing phenomenon, but a matter of degree. There are
several factors that could result in a Gram-positive organism staining Gram-negatively:
1. The method and techniques used. Overheating during heat fixation, over decolorization
with alcohol, and even too much washing with water between steps may result in Gram-
positive bacteria losing the crystal violet iodine complex.
2. The age of the culture. Cultures more than 24 hours old may lose their ability to retain the
crystal violet-iodine complex.
3. The organism itself. Some Gram-positive
Gram positive bacteria are more able to retain the crystal
violet-iodine
iodine complex than others. Therefore, one must use very precise techniques in Gram
staining and interpret the results with discretion.

Flow Chart
Observation

Demo link
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https://www.youtube.com/watch?v=AZS2wb7pMo4

Result