NAME: CECILIA MUKOTOTSI

REG NUMBER: H200313P

PROGRAMME: BIOTECHNOLOGY

COURSE NAME: CELL BIOLOGY

COURSE CODE: SBT 120

LECTURER: MR CHIROVA

FISRT PRACTICAL WRITE UP 3

Title:

Simple gram staining cells and the microscopic view of prokaryotic cells.

Abstract

The main purpose of the experiment was to be able to perform specialised staining
procedures in order to examine different properties of cells and to emphasize proper
techniques of how to handle cells. The preparations of films for staining was done using
bacterial epidermis. Gram staining was then performed where crystal violet solution, iodine
solution, ninety-five percent ethanol and safranin was used as stains. Water was used to
frequently wash was the slides after every staining stage. The slide was then examined under
the light microscope and the cells viewed where purple in colour, this means that there were
gram positive.

Introduction

A Gram stain is a test that checks for microorganisms at the site of a supposed contamination
or in certain body fluids, for example blood or urine. The sites which are usually checked
include the throat, lungs, and genitals and also in skin wounds. There are two main
typesof bacterial contagions which are Gram-positive and Gram-negative. There are two
types are identified based on how the bacteria responds to the Gram stain. When the bacteria
combines with the stains in a sample, during examination the bacteria will either stay purple
or turn pink. Thick layer of peptidoglycan in the cell that retains the primary stain which is
the crystal violet. Gram negative cells have a thinner peptidoglycan layer that allows the
crystal violet to remove the addition of ethanol, the gram negative are stained pink or red to
indicate the last indicate the colour of the last counterstain which is the safranin ( EL-Grarnal,
2009).

Some types of infections of gram negative are salmonella, pneumonia, urinary tract infections
and gonorrhoea. In gram positive some types of infections are toxic shock and strep
infections. The gram stain method is usually used to check the presence of bacterial infection
in human body and if it happens to show the infections the test will show if the infection is
gram positive or gram negative type. The infections can be checked on wound sample, blood
test, urinate test, throat culture, sputum culture samples. The gram stain can also be used in
diagnose of fungal infections (Colco, 2005).

According to Beveridge, (2001), for gram staining method to be done first, the specimen slide
is first mounted, fixed and a smear is then mad. Gram staining has four main part procedure
where different types of dyes are applied to make the bacterial cell to stand out against its
background. The slide is placed in a slide holder or a rack and it is flooded with the primary
dye, crystal violet for about a minute. The slide is then washed using water and the specimen
slide should have a purple when observed with naked eyes. The slide is flooded with iodine
solution whih acts as a mordant and after a minute it is washed again using water, the coliur
of the specimen should remain with the same colour purple. In stage three the specimen is
flooded with the decolorizer, ethanol, the decoloriser should not be too much or too little as
this affects the results in false gram positive or gram negative. The final stage is when th
specimen slide is flooded with the counterstain, safranin which is pink in colour and then the
slide is again washed with water.

The specimen slide is then examined using a light microscope under oil emmersion, the
results viewed are then explained according to shape, colour and arrangement as all of these
varies from different bacterial slides. The purple colour indicates that it is gram positive and
the pink colour means that it is the gram negative. Some of the shapes of the bacteria are, rod
shaped which is called bacillus and coccus which is spherical, spiral or curved in shape. The
slides are also described by their arrangement as bacteria are either singly, bunches or
branching (Leboff, (2014) .

The gram stain according to the shape and arrangement are specific for bacteria like
staphylococcus epidermis are always gram positive, cocci and in bunches , Escherichia coli
are always gram negative and rod shaped (Bailey, (2007).

Aims and Observations

To be able to perform specialised staining procedures in order to examine different properties

of cells and to reinforce proper technique for handling of cells.
Methodology

A clean slide was obtained and drawn a circle on it which was approximately one comma five
centi-meters in diameter and the slide was turned over. Using a loop a drop of water was
added to the circle on the slide and aseptically a small quantity of culture from thebacteria
culture plate provided was removed and mixed with the water to make a smooth suspension.
The suspension was allowed to dry and when dried, it was then fixed by passing it for
several times through a Bunsen burner flame until the slide was warm. The smear was
flooded with crystal violet for one minute and washed using tape water for some seconds.the
water was removed from the slide by tapping gently on a paper towel. Gram iodine solution
was added to the slide for one minute and then washed gently again. The slide was then
decolorised using ninety-five percent ethanol until no more colour was removed and it was
rinsed gently with water. The water was removed using paper towel. The slide was
counterstained using safranin for about one hour. The slide was then washed, air dried on a
paper towel and examined under oil emmersion.

Results

Fig1.1 showing the bacterial cells under light microscope

The bacterial cell shows a gram positive, which was purple in colour. They were cocci in
shape as they appeared round and spherical. The cells were arranged in bunches and the cells
also appeared to have same diameter. The microscope appearance was in grape like clusters
and the number of cells in clusters varied and the total number of cells were too many to
count.

Discussion

Our results proved that the experiment was carried out successfully as correct amount of dyes
and the time needed to flood and wash the dyes was maintained precisely. There are two
types of bacterial cells, which are gram positive and gram negative. The bacteria observed
was the gram positive, this was showed by the colour purple which was retained from the
primary die. As shown by the results the cocci shape of the bacterial shape and the bunching
in arrangement indicates that the bacterial cell is from staphylococcus epidermis group as
they are always found in gram positive, cocci and in bunches, according to shape, colour and
arrangement (Fedtke, 2007).

Conclusion

According to the aims and objectives of the experiment and the results obtained it can be
concluded that the students were able to perform specialised staining procedures and
examined different properties of bacterial cells. The students were able to differentiate two
different types of bacterial cells when gram stain is applied to then which are gram positive
and gram negative. They were also able to describe the gram positive or gram negative
bacterial cells according to their colour, shape and arrangement.
References

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