Gram Staining:
principle:
Gram
staining is a common staining method or technique used to differentiate two
large groups of bacteria based on their different cell wall constituents. The
Gram stain procedure distinguishes between Gram positive and Gram negative
groups by coloring these cells red or violet. Gram positive bacteria stain
violet due to the presence of a thick layer of peptidoglycan in their cell
walls, which retains the crystal violet these cells are stained with.
Alternatively, Gram negative bacteria stain red, which is attributed to a thinner
peptidoglycan wall, which does not retain the crystal violet during the
decoloring process.
When the bacteria is stained with primary stain Crystal Violet and fixed
by the mordant, some of the bacteria are able to retain the primary stain and
some are decolorized by alcohol. The cell walls of gram positive bacteria have
a thick layer of protein-sugar complexes called peptidoglycan and lipid content
is low. Decolorizing the cell causes this thick cell wall to dehydrate and
shrink, which closes the pores in the cell wall and prevents the stain from
exiting the cell. So the ethanol cannot remove the Crystal Violet-Iodine
complex that is bound to the thick layer of peptidoglycan of gram positive
bacteria and appears blue or purple in colour. In case of gram negative
bacteria, cell wall also takes up the CV-Iodine complex but due to the thin
layer of peptidoglycan and thick outer layer which is formed of lipids,
CV-Iodine complex gets washed off. When they are exposed to
alcohol, decolorizer dissolves the lipids in the cell walls, which allows
the crystal violet-iodine complex to leach out of the cells. Then when again
stained with safranin, they take the stain and appears red in color.
Reagents Used in Gram Staining:
- Crystal
Violet, the primary stain
- Iodine,
the mordant
- A
decolorizer made of acetone and alcohol (95%)
- Safranin,
the counterstain
Procedure of Gram Staining:
1.Take a clean, grease free
slide.
2.Prepare the smear of
suspension on the clean slide with a loopful of sample.Air dry and heat fix:
The first consideration is the correct preparation of the smear. Make a
thin film of the material on a clean glass slide, using a sterile loop or swab
for viscous specimens. Air dry, then heat fix the slide by passing it several
times through a flame (the slide should not become too hot to touch). Failure
to follow these directions may cause staining artifacts and disrupt the normal
morphology of bacteria and cells.
3.Crystal Violet was poured
and kept for about 30 seconds to 1 minutes and rinse with water.
4. Flood the gram’s iodine for
1 minute and wash with water.
5. Then ,wash with 95% alcohol
or acetone for about 10-20 seconds and rinse with water.
6. Add safranin for
about 1 minute and wash with water.
7. Air dry, Blot dry and
Observe under Microscope.
Results:
As
shown below, organisms that retain the violet-iodine complexes after washing in
ethanol stain purple and are termed Gram-positive, those that lose this complex
stain red from the safranin counter stain are termed Gramnegative
REFERENCE:
1. Charlton T. Lewis,
Charles Short, A Latin Dictionary s.v. "gramma" Archived 2015-07-17 at the Wayback Machine, 1879
2. Knorr, Wilbur R. (1996).
"Carmen de ponderibus et mensuris". In Hornblower, Simon; Spawforth,
Antony (eds.). The Oxford Classical Dictionary (3rd ed.). Oxford: Oxford
University Press. p. 292. ISBN 019866172X.
3. Henry George Liddell.
Robert Scott. A Greek-English Lexicon (revised
and augmented edition, Oxford, 1940) s.v. γράμμα Archived 2015-07-17 at the Wayback Machine, citing the 10th-century work Geoponica and a 4th-century papyrus edited in L.
Mitteis, Griechische Urkunden der Papyrussammlung zu Leipzig, vol.
i (1906), 62 ii 27.
4. "System of
Measurement Units - Engineering and Technology History Wiki". ethw.org. Archived from the original on 29 April 2018.
Retrieved 29 April 2018.
5. "Circulating Coin Designs". Japan Mint. Archived
from the original on 18 September 20
Tags
Microbiology