II.

DIFFERENTIAL STAINING


a) The Gram Stain

INTRODUCTION:

The Gram stain which is the most broadly used differential stain in
bacteriology, is named after Christian Gram who was the discoverer in 1884. There
are two groups of bacteria : Gram negative and Gram positive which differ in the cell
walls constituents.

Due to the thick layer of peptidoglycan layer in their cell walls, Gram positive
bacteria take up the crystal violet stain used in the Gram staining process. Teichoic
acids are found in the cell wall of Gram-posituve bacteria. Polymers of glycerol,
phosphates and the sugar alcohol ribitol make up the teichoic acids. Teichoic acids
are divided into ribitol teichoic acid and glycerol teichoic acid. Major teichoic acid
found in Gram-positive bacteria is the lipoteichoic acid which have a lipid component in
the cell membrane that can assist in anchoring the peptidoglycan. The alcohol rinse does not
remove the crystal violet, which masks the added red safranin dye.
On the other hand, Gram-negative bacteria do not retain the the crystal violet
stain used in the Gram staining process. A thin peptidoglycan layer is found on the
cell walls of the Gram-negative bacteria. The peptidoglycan layer is sandwiched
between inner plasma membrane and a bacterial outer membrane. There are no
teichoic acids found in the cell walls of Gram-negative bacteria. On the cell wall of
Gram-negative bacteria, there is an outer layer which consists of lipopolysaccharide,
lipoprotein and phospholipid. An alcohol solution removes the outer lipid-based
membrane of Gram-negative bacteria. By dissolving away the previously applied
crystal violet , the alcohol solution also decolourises the exposed peptidoglycan
layer.



METHODS:

1. A heat-fixed smear is prepared of the Escherichia coli, Bacillus subtilis,
Staphylococcus aureus, and Kiebsiella aerogenes.

2. The slide is placed on a staining rack and the smear is covered with cystal
violet for 1 minute.

3. The stained is washed off gently with tap water.

4. The smear is then covered with iodine and the smear is left for 1 minute which
is then washed off with tap water.

5. The slide is decolourised with alcohol by tilting it over the sink and dripping
the solution on to the slide until the washings are practically colourless.

6. After that, the slide is washed well with tap water.

7. The smear is counterstained with safranine for 1 minute.

8. The slide is washed and blotted dry gently.

9. The slide is then examined under oil immersion with maximum light in order to
distinguish colours.

10. The observations are recorded.



RESULTS:

Types of microorganisms Description

Escherichia coli


















Under 100x magnification

In the Gram staining process, E.Coli is
stained red and it is in rod-shaped or
Bacillus

E.Coli is arranged in a chain form and
singular rod.

Therefore, E.Coli is categorized in Gram-
negative bacteria.

Bacillus subtilis







In the Gram staining process, Bacillus
subtilis is stained purple and it is in long
rod-shaped or Bacillus.

The Bacillus subtilis is arranged in singular
rod.













Under 100x magnification
Therefore, Bacillus subtilis is categorized
in Gram-positive bacteria.

Types of microorganisms Description

Staphylococcus aureus



















Under 100x magnification

In the Gram staining process,
Staphylococcus aureus is stained purple
and it is spherical shape or coccus.

Staphylococcus aureus is arranged in
bundles.

Therefore, Staphylococcus aureus is
categorized in Gram-positive bacteria.

Klebsiella aerogenes











In the Gram staining process, Kiebsiella
aerogenes is stained red and it is in
coccusbacilli shape which is in small size.

Kiebsiella aerogenes is arranged singly.

Therefore, Kiebsiella aerogenes is
categorized in Gram-negative bacteria.








Under 100x magnification


Due to the thicker peptidoglycan layer and lower lipid content in the cell walls,
Bacillus subtilis and Staphylococcus aureus are categorised under Gram-positive
bacteria as these bacteria retained the crystal violet stain at the end of the
experiment. The alcohol solution is not able to decolourise the crystal violet stain on
these two groups of bacteria. It is because the cell walls trap the insoluble crystal
violet-iodine complex which resist the decolourisation. Counterstain which is the
safranine has no effect on the Gram-positive bacteria as the bacteria are stained
purple. Dehydrating the cell wall, the alcohol causes the pores to close as the cell
wall shrinks during this process.Thus, the diffusion of the crystal violet-iodine
complex is blocked and the microorganisms remain stained purple.
On the other side, Escherichia coli and Klebsiella aerogenes are Gram-negative
bacteria based on the observation as the bacteria are stained red under microscope.
The thin cell walls of Gram-negative bacteria are not able to retain the crystal violet-
iodine complex. The alcohol solution is able to decoloursie the crystal violet-iodine
stain on the smear. When the counterstain is carried out, the Gram-negative bacteria
is now stained red as it is colourless after being decolourised by the alcohol solution.


QUESTIONS:

a. What colour would you expect Staphylococcus aureus to be if the iodine step was
omitted in the gram staining procedure?

If the iodine step was omitted in the gram staining procedure, the Staphylococcus
aureus will be stained red in colour. This is because no crystal violet-iodine complex
will be formed as there is no iodine molecule that will react with crystal violet
molecule. Crystal violet-iodine complex gives the bacteria purple colour and without
the complex, alcohol solution will decolourise the crystal violet stain and the
Staphylococcus aureus will be stained red after safranine is applied.

b. Why would methylene blue not work just as well as safranine for counter staining
in the gram staining procedure?

The methylene blue does not work just as well as safranine for counter staining in
the gram staining procedure because the Gram- negative bacteria will be stained
blue in colour instead of red in colour is safranine is used. Then, it will be very
difficult when observing the bacteria under microscope as the blue and the purple
colour from crystal violet-iodine complex do not show significant difference. And,
distinguishing Gram-positive bacteria and Gram-negative bacteria will be tougher.
Thus, safranine is more suitable option to be used in counterstaining.