THE GRAM STAIN
Introduction
Gram's Stain is a widely used method of
staining bacteria as an aid to their
identification. It was originally devised by
Hans Christian Joachim Gram, a Danish
doctor.
Gram's stain differentiates between two
major cell wall types. Bacterial species with
walls containing small amounts of
peptidoglycan and, characteristically,
lipopolysaccharide, are Gram-negative
whereas bacteria with walls containing
relatively large amounts of peptidoglycan and
no lipopolysaccharide are Gram-positive.
It's a mystery
Although it may seem strange, the reason
why bacteria with these two major types of
bacteria cell walls react differently with
Gram's stain appears to be unconnected
with the wall structure itself. The exact
mechanism of the staining reaction is not
fully understood, however, this does not
detract from its usefullness.
The Gram staining method
1. A small sample of a
bacterial culture is removed
from a culture. Using a sterile
inoculating loop, remove a
small colony from your agar
plate. Only a very small
amount of culture is needed; a
visual detection of the culture
on an inoculation loop already
indicates that too much is taken.
2. Place a drop of water onto a
glass slide. The bacterial
suspension is then smeared
onto the glass slide. Spread the
culture with an inoculation loop
to an even thin film over a
circle of 1.5 cm in diameter,
approximately the size of a
dime.
3. Air-dry the culture and fix it
or over a gentle flame, while
moving the slide in a circular
fashion to avoid localized
overheating. The applied heat
helps the cell adhesion on the
glass slide to make possible
the subsequent rinsing of the
smear with water without a
significant loss of the culture. Care must be
taken not to overheat which will char the
cells.
4. Once cool, the slide is
transferred to a support over a
sink and flooded with a stain
called Crystal Violet (a dye
consisting of a methyl
derivative of pararosaniline).
The stain is left on the slide for
about 1 minute. This stains all
the bacteria on the slide a dark purple color.
Note, this stain will not penetrate the waxy
cell walls of some bacteria eg mycobacteria
5. The Crystal Violet is gently
washed off the slide with
running water
6. The bacterial smear is then
treated with Gram Iodine
solution. Add the iodine
solution on the smear, enough
to cover the fixed culture. This
iodine solution reacts with the
Crystal Violet turning it a very
dark shade of blue. It also
causes it to be retained by certain types of
bacteria in a way which is not really
understood.
7. After about 30 seconds the
slide is gently rinsed with
ethyl alcohol which causes
the dye-iodine complex to be
washed out of some bacteria
but not others. This is called
decolorization. Add a few drops
of decolorizer so the solution
trickles down the slide.
If we now looked at the smear down a
microscope, the bacteria which had retained the
Crystal Violet-iodine complex will appear blueblack. These are called Gram-positive. However
we would not be able to see those which had
lost the dye-iodine complex which are called
Gram-negative. The final step in the Gram stain
method is, therefore, to stain the Gram-negative
cells so they can be seen.
8. This is achieved by treating
the smear with a compound
which stains the Gram-negative
cells a color which contrasts
markedly with the blue-black
color of the Gram-positive cells.
The stain common used for this
is either Safrinin or Fuchsin,
both of which are red. These are called
counterstains. Bacteria in the smear which are
Gram-positive are unaffected by the
counterstain.
9. The counter stain is left on
the smear for about 30-60
seconds and then gently rinsed
away with running water.
10. After the counterstain has
been rinsed off, the slide is
placed between some
absorbent paper and the excess
water gently blotted off. Care
must be taken not to rub the
slide with the blotting paper
because this would remove the
adhering bacteria.
11. The slide is gently warmed
to drive off any residual
moisture and then a drop of
immersion oil is placed on
the stained bacterial smear.
This helps transmit light
through the specimen directly
to the high-powered
microscope lens.
12. The slide is then placed on
a microscope stage and the oilimmersion lens (100x) lowered
into the immersion oil. Highpowered lenses are required
because bacteria are very
small.
The Results
Typical Gram-positive bacteria
Gram positive
1.
Staphylococci such as Staphylococcus
epidermidis and Staphylococcus aureus
which is a common cause of boils
2.
Streptococci such as the many species of
oral streptococci, Streptococcus
pyogenes which causes many a sore
throat and scarlet fever and
Streptococcus pneumoniae which causes
lobar pneumonia
3.
Clostridia such as Clostridium tetani
which cause tetanus (lockjaw)
4.
Actinomyces such as Actinomyces
odontolyticus which is found in mouths
5.
Species of the genus Bacillus such as
Bacillus subtilis which are common
microbes living in soil
Staphylococcus epidermidis
Typical Gram-negative bacteria
The bacilli that cause
1. Whooping cough, Bordetella pertussis
Gram negative
2. Typhoid, Salmonella typhi
3. Cholera, Vibrio cholerae
4. The normally benign, ubiquitous, gutdwelling Escherichia coli
Escherichia coli
Generally cocci are Gram-positive but there
are exceptions. The most significant from a
clinical point of view is the gonococcus,
Neisseria gonorrhoea which typically appears
as a Gram-negative diplococcus looking very
much like a pair of kidney bean.
And Finally
SUMMARY
It is important to recognize that not all
species of bacteria can be usefully stained by
Gram's method. Some species are Gram
variable and some what is called Gram
indeterminant. It is also worth mentioning
that the growth conditions may also affect a
bacterial species Gram reaction. For example,
Gram positive cells growing in batch culture
which are into the decline phase of the
growth cycle often show numerous Gram
negative cells present.
1.
Although apparently unconnected with
the bacterial cell wall structure, Gram's
stain differentiates between two major
cell wall types. Bacterial species with
walls containing small amounts of
peptidoglycan and, characteristically,
lipopolysaccharide, are Gram-negative
whereas bacteria with walls containing
relatively large amounts of peptidoglycan
and no lipopolysaccharide are Grampositive.
2.
Examples of Gram-negative bacteria are
Escherichia coli, Salmonella typhi, Vibrio
cholerae and Bordetella pertussis
3.
Examples of Gram-positive bacteria are
Staphylococcus epidermidis,
Streptococcus pyogenes, Actinomyces
odontolyticus and Clostridium tetani
4.
Not all bacteria can be stained by Gram's
method, the best-known exception
belong to the genus Mycobacterium
which have waxy cell walls.
Another major exception is the genus
Mycobacterium which includes such important
human pathogens as Mycobacterium
tuberculosis (TB) and Mycobacterium leprae
(leprosy). Bacteria such as these have very
different cell walls which contain a great deal
of waxy material, although they still contain
peptidoglycan. This waxy material prevents
stain penetration rendering the cells invisible.
Special methods have been devised to stain
these bacteria involving the use heat to allow
the stain to permeate the cell walls. Once
cooled even dilute acid fails to remove it
hence the name "acid-fast" to describe these
species of bacteria.
Since these acid-fast bacteria are not stained
by Gram's method they have been described
as "Gram-negative". Strictly speaking this is
true but it can be misleading to refer to them
this way.
Footnote
The division of all life into prokaryotes and
eukaryotes was challenged by Carl Woese in
1977. He pioneered the use of 16S ribosomal
RNA in the study of taxonomy which was a
major breakthrough in our understanding of
the relationships between all forms of life and
used this technique to reveal major
differences in prokaryotic micro-organisms.
In 1990 Woese proposed that the existing
Kingdom of Prokaryotes be divided into
Archaea and Bacteria. Based on his research,
which showed that these two groups evolved
quite independently from a common
ancestor, the consensus view now is that the
Kingdoms called Prokaryota and Eukaryota
be replaced by three domains (sometimes
called Superkingdoms or Empires) called
Archaea, Bacteria and Eukarya.