SCHOOL OF INDUSTRIAL SCIENCES AND TECHNOLOGY

DEPARTMENT OF BIOTECHNOLOGY

PRACTICAL WRITE UP 2
COURSE TITLE : MICROBIOLOGY

COURSE CODE : SBT120

LECTURER : MR CHIROVA

STUDENT NAME : NAMAKANDO DUTTON

STUDENT NUMBER : H180036J

Simple and Gramm staining of bacteria

Abstract

An experiment was conducted in which a slide of bacteria was made and examined. The bacteria
used was Staphylococcus aureus. The bacteria was placed onto a glass stain with a loop that was
sterilised by heating the inoculating loop until it was red hot. The slide was then fixated and stained
with Crystal violet and Grams iodine. The slide was then decolourised with 95% alcohol and then
counterstained by Safranin. The slide was washed with water and dried. The prepared slide was
then examined under a microscope at a magnification of x100 in an oil immersion. Round, purple,
grape-like clusters of cells were observed under the microscope

Introduction

All cells in organisms are very small and cannot be seen with the naked eye. A microscope is
therefore used in order to examine the cells of organisms. According to Iwasa and Marshall (2016),
“Microscopes are instruments that produce an enlarged image of an object.” The microscope can
use different magnifying factors. Larger objects can be examined under a magnification of x10,
whereas smaller objects will need a larger magnification of x100.

A light microscope makes use of a light located in the base of the microscope. The light source
illuminates the specimen that is to be examined. The condenser lens focuses the light rays on the
specimen and these light rays are then collected by the objective lens. There are two rays of light
that enter the objective lens: those that are altered by the specimen and those that are not. The
unaltered light rays enter the objective lens directly. The altered light rays enter through the
specimen and for the image. The objective lens then focuses the light rays in order for the image to
be enlarged. The ocular lens uses the image formed by the objective lens as an object to form a
larger image. The focusing knob on a microscope can be turned to change the relative distance
between the specimen and the objective lens. This allows the image to be focused on the plane of
the retina. The total magnification is a product of the of the objective and ocular lens (Iwasa and
Marshall, 2016). The working distance is the distance between the lens of the microscope and the
prepared glass slide.

Iwasa and Marshall (2016) states that, “the resolving power of a microscope can be defined in
terms of the ability to see two neighbouring points in the visual field as two distinct entities.” From
the definition it is seen that the better the resolution of a microscope the clearer and more distinct
 the image will be. An image with high resolution will be easier to see and analyse. The resolution
can be affected by the bending of light. This can be avoided by covering the cover slip with an oil
emmersion that has the same refractive index as that of the cover slip and the lens.

Bacteria can be identified by several ways. All bacterial cells of the same type have the same
properties and shape and these are used to help identify the bacteria. Some bacteria are round, rod
shaped or linear. The bacteria can also be identified by how many cells tend to be present per
cluster. Bacteria of the same type will absorb the same dyes and therefore be the same colour when
stained.

In 1884 it was discovered that pararosanilin dyes and iodine form insoluble compounds and a
staining method was then derived. It was also discovered that some products were soluble in
alcohol while others were insoluble. In Gramm staining Crystal violet the first stain to be used and
is followed by an iodine solution. The two solutions form a complex that cannot be removed by
alcohol. The iodine also increases the interactions of the cell and the dye thereby increasing the
intensity of the stain (Prescott, 2002). The compounds that are insoluble in alcohol are known as
Gram positive. The alcohol acts a decolouriser (Bartholomew and Mittwer,1952). In order to make
Gram negative compounds visible again they must be counterstained. Gram negative compounds
appear pink when viewed under a microscope. A large number of organisms are Gram negative and
cannot keep the violet stain in the presence of alcohol (Huck and Conn). The staining of the
specimen adds colour to the slide and makes it easier to see the different regions of the cells as they
are darkened and therefore more visible and easy to distinguish. The different parts of the cell
absorb different amounts of the stain applied to the specimen and so they have different shades.
This makes the cell easier to analyse.

Aims and objectives

Aim – to view bacteria under a microscope

- to identify the bacteria under the microscope

Objective – to perform specialised staining procedures in order to examine different properties of

bacteria
- to reinforce proper techniques for handling microorganisms
 Method and materials

Materials

Stains: Crystal violet

Safranin
Gram’s iodine
95% ethanol
Bacteria culture : Staphylococcus aureus
Microscope

Exercise 1: Preparation of Films for Staining

1. Obtain a clean slide and draw a circle on it approximately 1.5 cm in diameter. Turn the slide over.
2. Using a loop add a drop of water to the circle on the slide. Aseptically remove a small
quantity of culture from the plates provided and mix with the water to make a smooth suspension.
3. Allow the suspension to air dry. When dry, the film should be only faintly visible; a thick
opaque film is useless.
4. The only fixation required is to pass the slide several times (maximum 10) through the
bunsen burner flame until the slide is warm but not too hot.

Exercise 2: Gram Staining

1. Flood the smear with crystal violet solution for 1 min.

2. Gently wash with tap water for 2-3 seconds and remove the water by tapping the slide gently
on paper towel.
3. Add Gram’s iodine solution to the slide for 1 min. Wash gently with tap water and remove
as above.
4. Decolourise with 95% ethanol by dripping ethanol on surface of slide until no more colour is
removed.
5. Rinse gently with water. If too much alcohol is added, the Gram-positive organisms may
become Gram-negative.
6. Remove the water after the last wash.
7. Counterstain the slide with safranin for 30 seconds - 1 minute.
8. Wash the slides with tap water, air dry on paper towel and examine under oil emmersion

Results

Round, purple, grapelike cells were observed under the microscope

Figure 1: Drawing of the Staphylococcus Aureus viewed under the microscope at a magnification
of x100

Discussion

The cells that were stained on the microscopic slide were Gram positive. This can be concluded
because the purple dye remained on the cells even after being rinsed in 95% alcohol.

There are different things that could have affected the results that were obtained. If the layer of the
culture of the Stapylococcus Aureus used was too thick it would have affected the accuracy of the
slide. When the layer is too thick, the specimen is difficult to observe because the cells are
overlapping and are on top of each other making the visibility of one specific cell difficult.
The specimen is also best observed when it is moist. The specimen dries up as time goes on. This
means that the specimen must be observed when it is still fresh. It must be put under the
microscope immediately after it is prepared.

The time the culture was incubated for also affects what is seen on the slide. Cultures of different
ages absorb the stain at different concentrations. Older cells may appear to be gram negative when
they are actually gram positive.

The amount of alcohol used to rinse the slide must be moderate. If too much alcohol is used then it
damages the cell walls of the organism being observed. The specimen that is observed is therefore
not accurate.

Conclusion
The specimen that was on the slide was visible and therefor was gram positive. The specimen was
identified as being Staphylococcus Auerus.
REFERENCES

1. Bartholemew. J. W and Mittwer. T., 1952, The Gram Stain, Department of

Bacteriology, University of South California, Los Angeles
2. Hucker. G. J. and Conn. H. J., 1923, Methods of Gram Staining, New York
Agricultural Experiment Station, New York
3. Iwasa, J. and Marshall, W., 2016, Karp’s Cell and Molecular Biology, 8th Edition,
Wiley, USA
4. Prescott. L. M., Harley. J. P. and Klein. D. A., 2002, Microbiology, 5th Edition,
McGraw-Hill
5.