THE STAINING OF MICROORGANISMS

Bacterial morphology may be examined in two ways:

1. By observing the living, unstained organisms as is done in demonstrating

bacterial motility.
2. By observing dead cells stained with dyes.

Living bacteria are almost colourless and do not present sufficient contrast with
the water in which they are suspended to be clearly visible. Staining the
organism will make them contrast in colour with their surrounding, so they are
more readily visible. Certain stains can also be used to identify certain internal
structure of the cell, which would otherwise pass unseen. Further, in order to
use the oil immersion objective and obtain the greatest degree of magnification,
it is more convenient to use stained preparation than wet mounts.

A. DIRECT STAINING WITH BASIC DYES

When a staining procedure colors the cells present in a preparation, but leaves
the background colorless (appearing as white), it is called a direct stain. When
preparing cellular materials for direct staining it is important to remember that
morphological

features are most readily visible if the cells present are separated from one
another rather than being packed into a dense mass. Direct staining stained
bacteria with basic dye (methylene blue, carbol fuchsin, crystal violet,etc)
which its colouring power is in positive ion, and reacts with the negatively
charged cell. Thus, the bacterial cell will be stained with the basic dye's colour.
In other words, direct staining can be explained as staining the bacterial cell.
SIMPLE STAINING TECHNIQUES

Materials

1. 24 hours broth cultures of

a. E.coli
b. Staph. Aureus
2. 24 hours nutrient agar slants of
a. Bacillus subtilis
b. Pseudomonas aeruginosa
3. Slides
4. Inoculating loop
5. Dye solutions
a. Crystal violet
b. Methylene blue
c. Carbol fuchsin
6.Test tubes

Procedure

1. Some saliva were collected in the test tube provided.

2. The film for staining were prepared.
3. The film were flooded with approximately 5 drops of stain and stains were
allowed to react: Methylene blue-30 seconds, crystal violet-10 seconds,
carbol fuchsin-5 seconds.
4. The stain were washed off with tap water, excess water were blotted off
and stained film were dried over bunsen flame.
5. The slide were examined and images seen under microscope were drawn
and results were recorded.

Diccussion

I) Staphylococcus aureus

The shape of Staphylococcus aureus is spherical or in other words, they

were seen in clusters of cocci.
The size of this bacteria is small.
Staphylococcus aureus is arranged like a bunch of grapes.

III) Escherichia coli

Escherichia coli is a rod shaped bacterium.

It is short and it has individual arrangement.
It is not clustered together like Staphylococcus aureus.

Simple staining is a method that is used in microscopy to enhance the

visualization of microscopic images. Stains and dyes are the two major
chemicals that are often used in simple staining to accentuate the structure
and the morphology of the bacterial cell. In this experiment, methylene blue
solution was used to stain the bacterial smears. Why is methylene blue used to
stain the bacterial cells in this experiment? Since methylene blue is a basic dye,
it has a high affinity for acidic substances. The presence of negatively charged
molecules in the cells causes it to be attracted to the positively charged dye.
 Due to the staining procedure, the morphology of the bacterial cell will
be clearly seen when examined under the oil immersion. From the observation,
we can infer that both Escherichia coli are rod shaped bacteria. However, we
can note the difference in their arrangment. Escherichia coli is arranged in an
individual manner with noticeable space between each cell. The bacteria
Staphylococcus aureus appears oval shaped, like a bunched grape like
clusters. It is also very small in size compared to the other three bacteria.

Conclusion

In a conclusion, the morphology of the bacteria were observed with the aid of
simple stain. Simple staining technique is the best method to observe the
morphology, size and the arrangement of bacteria.

Question
1. In preparing a slide from a bacterial colony growing on an agar medium,
why is it necessary to place only a minute portion of the colony on the
slide?
Only a minute portion necessary to be taken from an agar medium of
bacterial colony because the bacteria cells might overlap with each other if
it is taken too much

References
1. Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., & Riccardi, C.
(1991). A rapid and simple method for measuring thymocyte apoptosis by
propidium iodide staining and flow cytometry. Journal of immunological
methods, 139(2), 271-279.
2. Sidhu, K. S., Dhindsa, J. S., & Guraya, S. S. (1992). A simple staining
procedure for detecting the true acrosome reaction in buffalo (Bubalus
bubalis) spermatozoa. Biotechnic & histochemistry, 67(1), 35-39.
3. Kobus, H. J., Warrener, R. N., & Stoilovic, M. (1983). Two simple staining
procedures which improve the contrast and ridge detail of fingerprints
developed with Super Glue(cyanoacrylate ester). Forensic Science
International, 23(2-3), 233-240.