2. INTRODUCTION
Before discussing gram staining procedure I want to
discuss about the history of Microbiology in short.The
credit for having first observed and reported bacteria
belongs to ANTONY VAN LEEUWENHOEK , a draper in
Delft, Holland, whose hobby was grinding lenses and
observing diverse materials through them.
But the development of Microbiology as a scientific
discipline dates from LOUIS PASTEUR (1822-95).He
introduced the techniques of sterilisation and developed
the steam steriliser, hot air oven and autoclave.He also
established the differing growth needs of different
bacteria and contributed to the knowledge of Anthrax,
Chicken Cholera and Hydrophobia.
3. It was Pasteur who coined the term Vaccine. But that time
scientists found that bacteria do not show much structural
detail under light microscope due to lack of contrast.
This problem was solved by the discovery of staining
methods of microbes. Gram staining was devised by the
Danish histologist HANS CHRISTIAN GRAM in 1884 as
a method of staining bacteria in tissues. This is the most
important staining method in Bacteriology. It is used to
differentiate bacterial species into two broad groupes ,
Gram positive & Gram negative based on the physical
property of their cell wall.
4. TYPES OF STAINING TECHNIQUES
1.SIMPLE STAINS
Methylene blue or basic fuchsin are used.
They provide colour contrast but impart the same colour to all
bacteria.
2.NEGATIVE STAINING
Indian ink or nigrosin are used.
They produce uniformly coloured background against which
the unstained bacteria stand out in contrast.
Particularly useful in the demonstration of bacterial capsule
which do not take simple stain and also for spirochetes.
5. 3.DIFFERENTIAL STAINS
These stains impart different colours to different bacteria or bacterial
structures.
Primary staining is done by one dye
Counter staining is done by a different dye of contrasting colour.
Example---Gram stain and Ziehl-Neelsen stain.
4.IMPREGNATION METHOD
Silver impregnation method
Used for structures and cells too thin to be seen under the ordinary
microscope like spirochetes and bacterial flagella.
They may be rendered visible if they are thickened by impregnation of
silver on their surface
.Example—Fontana’s and Levaditi’s methods of staining
6. There are several other staining methods like--
ALBERT’S STAIN for volutine granules
GIEMSA’S STAIN for protozoa
LEISHMAN’S STAIN for protozoa in blood film
FLUROCHROME(AuramineO) STAIN for AFB
MALACHITE GREEN STAIN for spores
PERIODIC ACID-SCHIFF STAIN for fungi in tissue
SUDAN BLACK B STAIN for intracellular lipid of
bacteria
7. PROCEDURE OF GRAM STAINING
The Gram procedure requires the application of four
reagents in the following order:
1. A basic pararosaline dye such as crystal
violate,methyl violate or gentian violate
2.An aqueous solution of iodine
3.A decolourizing solvent, like acetone, alcohol or
aniline
4.A light red or pink counterstain,like basic fuchsin,
neutral red or safranine
8.
9. In the original method of Gram(1884) the smear was
stained with aniline-gentian violate,treated with
Lugol”s iodine, decolourised with absolute alcohol,
and counterstained with Bismarck brown.Later
modifications give better results
Crystal violate or methyl violate is used in the
concentration of 0.5-2%
Solution is facilitated if the dye is first dissolved in
alcohol,filtered through filter paper and added to the
water
10. However Gram positive staining can be strengthened by
addition of sodium bicarbonate or ammonium oxalate as in
the following solutions:
KOPELOFF & BEERMAN’S(1922) MODIFICATION:
Sol. A Methyl violate 6B 10 g
Distilled water 1 litre
Sol. B Sodium bicarbonate 50 g
Distilled water 1 litre
Mix 30 vol. of Sol A with 8 vol. of Sol B shortly before use.
Problem of this mixture is that it tends to precipitate
within a few days, so cannot be kept.
11. PRESTON & MORRELL’S(1962) MODIFICATION:
Crystal violate 20 g
Methylated spirit 200 ml
Ammonium oxalate, 1% in water 800 ml
It also strengthen gram positive staining.
Another one modification is-
JENSEN’S MODIFICATION: Here alcohol is used as a
decolourizer & weak neutral red as counterstain for examination
of gonococci & meningococci
IODINE SOLUTION(Gram’s iodine)-
Dissolve 20 g of Potassium iodide in 250 ml water & then add 10
g iodine.When iodine is dissolved, make upto 1 lt with water.
12. In KOPELOFF & BEERMAN’S modification iodine is
dissolved in NaOH sol. & then added distilled
water.The more alkaline sol. with NaOH is thought to
give stronger gram positive reaction.
DECOLOURIZER:
Acetone: it is the fastest & most specific
decolourizer.It is applied to smears for only 2-3 sec.
However the short period of exposure is difficult to
control when many slides are stained simultaneously.
13. Absolute alcohol(100% ethanol)-It acts more slowly
than acetone & should be applied for about 1 min.
Acetone-alcohol-It is a mixture of 1 vol. of acetone
with 1 vol. of 95% ethanol.It requires application for
about 10 sec.
Iodine-acetone-PRESTON & MORREL(1962) have
shown that addition of 0.35% iodine to acetone slows
its rate of decolourization & exposure can be
lengthened from 2 sec. to 30 sec.
14. But an irritating aerosol may be produced due to
splashing of iodine-acetone sol. into the sink.Acetone
quickly evaporates from the droplets & leaves airborne
droplet of iodine which irritate the eyes & nose.
To minimise this effect, iodine-acetone sol. should be
poured gently on to the slide through a wide nozzle &
splashing into the sink should be avoided.
COUNTERSTAIN-
1.Safranine-It is commonly used in 0.5% in distilled
water.
15. 2.Dilute carbol fuchsin-It gives the strongest red
staining.Colour may be so dark that some gram
negative bacteria may be difficult to distinguish from
gram positive one.
3.Basic fuchsin-It is weaker counterstain.It is
recommended for general use.
4.Neutral red-It is recommended for demonstrating
gonococci & other intracellular Gram negative
bacteria.
16. STAINING TECHNIQUE
1. Make a smear & dry thoroughly in cool air. Fix the
dried film by passing it briefly through a bunsen
flame.
2. Flood the slide with crystal violate sol. for upto 1
min.Wash off briefly with tap water & drain.
3.Flood the slide with gram’s iodine sol. & allow to act
as a mordant for about 1 min.Wash off with tap water
& drain.
17. STAINING TECHNIQUE
4.Decolourise the smear with acetone for 10-30 sec.
taking care not to overdecolourise & immediately wash
off with water.
5.Flood the slide with safranin sol. & counterstain for
about 30 sec., wash off with tap water, drain & blot dry
with filter paper & examine under oil immersion
objective.
21. Gram positive bacteria have a thick cell wall made of as
many as 40 sheets of peptidoglycan(50-90% of cell
envelope).
Whereas Gram negative bacteria have a thin cell wall
made of only one or two sheets of peptidoglycan(10%
of cell envelope).
Crystal violate(cv) dissociates in aqueous solution into
cv+ & chloride(cl-) ions.These ions penetrate through
the cell wall & cell membrane of both gram positive &
gram negative bacteria.
22. The cv+ ion interects with negatively charged components
of bacterial cells & stains the cells purple.Iodide(I-)
interects with cv+ & form large complexes of crystal violate
& iodine(CV-I) within the inner & outer layer of cell.
Iodine is a trapping agent that prevents the removal of CV-I
complex & therefore colour the cell.
When a decolourizer like alcohol or acetone is used, it
interects with the lipids of cell membrane.Gram negative
cell loses its outer lipopolysaccharide membrane & the
inner peptidoglycan layer is left exposed.
23. .
The CV-I complexes are washed from the gram
negative cell along with the outer membrane.
In contrast a gram positive cell becomes dehydrated
from an ethanol treatment.The large CV-I complexes
become trapped within gram positive cell due to
multilayered nature of its peptidoglycan.The
decolourization step is critical & must be timed
correctly.
CV stain is removed from both gram positive &
negative cells if decolourising agent is left on too long .
24. Conversely inadequate decolourization may cause all
cells to appear gram positive.
Examples of gram positive bacteria-
Staphylococcus,Streptococcus,Actinomycetes,Bacillus,
Corynebacteria,Clostridia,Listeria.
Example of gram negative bacteria-
Enterobacteriaceae,Pseudomonas,Neisseria,Vibrio etc.
Gram positive bacteria may sometimes appear gram
negative in ageing culture & when cell wall is
damaged.
25. Gram indeterminate bacteria or gram variable bacteria
do not respond predictably to gram staining.They tend
to stain unevenly, appearing partially gram positive &
pertially gram negative, or even unstained.
Examples-Micobacterium tuberculosis & leprae & also
Acinetobacter
Apart from bacteria, gram’s stain can also be used to
identify other microorganisms like Pneumosystis
jeroveci cysts, Strongyloides larvae,Toxoplasma gondii
trophozoites & Trichomonads etc.
26. A variety of artifacts can resemble infectious agents in
Gram’s stained smear.To overcome this problem a
useful maneuver is to stain another smear with
acridine orange; with this stain it can be established
whether the structure contain DNA & is, therefore,
biological.
In direct smear under-decolourization can be
monitored by observing the nuclei of inflammatory
cells; if they are not completely gram negative, the
smear has not been adequately decolourized.
27. The only recipe for detecting over-decolourization is
cross-checking the gram reaction & the morphology of
the bacteria.
