Bacteria culturing using mixed culture techniques to describe colony characteristics- Laboratory report. Makerere University (Ssusume Patrick)

1. Ssusume Patrick - DSTVE Makerere University
MAKERERE UNIVERSITY
COLLEGE OF EDUCATION AND EXTERNAL STUDIES
SCHOOL OF EDUCATION
FOUNDATION OF SCIENCE, TECHNICAL AND VOCATIONAL EDUCATION
NAME : SSUSUME PATRICK
REG No. : 20/U/1406
STUDENT’S No. : 2000701406
COURSE : BACHELORS OF SCIENCE WITH EDUCATION (BIOLOGICAL)
COURSE UNIT : MICROBIOLOGY, MYCOLOGY AND IMMUNOLOGY
(BACTERIOLOGY PRACTICAL)
COURSE CODE : BIO 3102
LECTURER : Dr. MULABI NICHOLAS ELIJAH
DATE : 22/11/2022
SEMISTER : 1
YEAR : 3
TITLE : CULTURING OF BACTERIA
AIM : TO CULTURE AND IDENTIFY DIFFERENT BACTERIAL
TYPES BASED ON THEIR MORPHOLOGICAL CHARACTERISTICS
USING MIXED CULTURE TECHNIQUES

2. Ssusume Patrick - DSTVE Makerere University
INTRODUCTION
Bacteria are microscopic, simple single-celled, ubiquitous and mostly free-living organisms that
constitute a large domain of prokaryotic organisms. The goal of the experiment is to culture and
identify different bacteria based on their morphological characteristics using mixed culture
techniques. Bacterial identification has significant importance in many different industries
including the pharmaceutical industry when considering microbial contamination. Microbial
contamination refers to the accidental introduction of infectious materials such as bacteria, yeast,
fungi or virus to the production line. The identification and characterization of bacteria is a vital
component in microbiology in both the scientific and clinical settings because they provide
valuable information regarding the microbial ecology of a specific environment, the metabolic
capabilities of a bacteria and their pathogenicity. This is useful in the clinical field in which
quick identification of bacteria provides information required to make a more accurate diagnosis,
prescribing an appropriate treatment and assessing the effectiveness of a treatment. By
identifying bacteria, sterilization of products can take place in the hope of removing pathogens
and reducing the risk of contamination. By inspecting for microbial contamination in products, it
ensures quality control as there will not be any damage in the way of chemical degradation,
which can lead to the loss efficacy as well as toxic side effects and medicine spoilage, all of
which can be detrimental to a patient’s life. Analytical techniques for example; morphological,
biochemical and genetical sequencing tests can be performed in order to identify different types
of microorganisms. Currently the identification of unknown bacteria by using biochemical and
physiological tests is considered for the most part obsolete because it is a process that can be
both labor intensive and time consuming carrying on days to weeks. Additionally, the test results
are never guaranteed to be completely accurate since bacteria mutate constantly to adapt to their
environments thus; they occasionally express genes that change a key biochemical or
morphological characteristics rendering this technique inadequate. In clinical setting, in which
speed is paramount, biochemical tests coupled with other immunological techniques are often the
preferred way to quickly assess whether a known specific bacterium is present or absent in a
sample. By sourcing the microbe’s identity, the growth and metabolism can be investigated with
the aim of finding an appropriate way to prevent it from growing any further and further
elimination; this is usually by recognizing the antibiotic which can destroy the cell membrane or
the function of the particular pathogen. In this experiment, mixed culture technique is used, that

3. Ssusume Patrick - DSTVE Makerere University
is; an inoculum expected to contain different bacterial strains picked anywhere is used owing to
their ubiquitous nature
MATERIALS USED
Nutrient agar Conical flask (250ml)
Microscope Beaker (100ml)
Glass slides and cover slips Inoculating wire loop
Weighing scale 70% ethanol
Water bath Petridish with cover (glass)
Autoclave Boiling tube
Cotton wool Stirring rod
Aluminium foil Methylene blue stain
PROCEDURE
Standardization of the medium
Nutrient agar (5g) was weighed and dissolved in 100ml of distilled water in a beaker. The
mixture was heated in a water bath while stirring to attain proper dissolution. The mixture in the
beaker was poured into a conical flask and tightly plugged with cotton wool. The conical flask
was covered up to the neck with aluminium foil (sheet)
Sterilization of culture medium and equipment
The petridishes and boiling tubes were covered with aluminium foil, the materials were sterilized
in an autoclave at 120o
C for 20 minutes to kill all pre-existing microbes.
Pour plating and slanting
The bench was swapped with 70% ethanol using cotton wool. The mouth of the conical flask
was swept in a flame to keep all floating microbes out of the culture medium. Nutrient agar was

4. Ssusume Patrick - DSTVE Makerere University
rapidly poured into the petridish opened at 450
angle. Nutrient agar was again poured in the
boiling tube held in a slanting position at an angle of about 45o
, the boiling tube and the petridish
containing the nutrient agar were covered immediately and the culture was left to solidify.
Inoculation of the sterilized culture medium
Dust (inoculum) was picked from a nearby window and the medium in petridish was touched
with dusty fingers and covered with a glass cover. This was as well done for the boiling tube.
The petridish and boiling tube were labeled with the source of the bacteria.
Incubation
The boiling tube and petridish (upside down) were placed in an incubator set at 38o
C for 3 days
for the microbes to reproduce.
Inspection of the plate and slant
After three days, the incubator was opened to check for the presence of bacterial colonies that
can be viewed with unaided eyes.
Microscopic techniques
Part of one bacterial colony was wiped with a sterilized inoculation wire loop and a thin smear
was made on a glass slide. The slide was placed on a staining rack over a sink and the smear was
flooded with methylene blue. The slide was left to stand for three minutes, the slide was gently
washed with tap water and the wet side blotted with a filter paper. The slide was covered with a
cover slip, an immersion oil drop applied and the slide was observed under high power of a
microscope.
Safe dumping of cultures
The used cultures and all used apparatus were collected in an autoclave and sterilized at a
temperature of 120o
C to kill all the bacteria. The apparatus were then removed and cleaned while
the agar was disposed-off.

5. Ssusume Patrick - DSTVE Makerere University
OBSERVATIONS
After heating, the nutrient agar mixture appeared turbid and yellowish in colour. After the three
days of incubation, bacterial colonies were observed at every point of nutrient agar that was
touched with dusty fingers. The colonies were visible with unaided eyes.
Appearance of the bacterial colonies growing on nutrient agar
Appearance of bacterial cells as observed under high power of a light microscope.

6. Ssusume Patrick - DSTVE Makerere University
DISCUSSION
Colony morphology and nature of bacterial colonies
The colonies appeared as yellow patches of different sizes in the petridish, irregular in shape,
with translucent opacity and flat elevation; they were larger than 1mm (isolated colonies) with an
undulate margin and a flat smooth surface. Growth on agar was abundant with no pigmentation.
Majority of the bacterial colonies were combined forming patches larger than 1cm. This
indicated presence of bacterial cells in the dust (inoculum) which were able to reproduce in
presence of adequate nutrients, favourable PH of the agar medium and favourable temperature of
the incubator (38o
C) to form bacterial colonies.
Nature of individual cells
On observation under high power the microscope, it was observed that some bacterial cells
stained blue, some yellow and others did not stain with methylene blue; this indicated presence
of different bacterial strains in the inoculum, some had thick peptidoglycan layer and therefore
were able to trap the train and were stained while those that were not stained due to the high lipid
content of their cell walls that does not facilitate trapping of the stain.
Bacterial cells of different shapes were observed; some were spherical and occurred singly
(monococcus) and others were rod shaped and occurred singly (monobacillus), some cells were
oval shaped (coccobacillus). This implies that there were mixed bacterial strains in the inoculum.
There were no fungal hyphae observed indicating that the dust was free of fungal
contaminations, this because the inoculum (dust) used was dry and most fungi grow in moist
places.

7. Ssusume Patrick - DSTVE Makerere University
CONCLUSION
In this experiment, unknown bacteria types were cultured using mixed culture techniques and
bacteria of different morphology, staining properties and colony characteristics were identified as
observed under high power of a microscope. Steam sterilization was employed using an
autoclave. The type of bacteria cultured were mesophilic since incubation was done at 38o
C. The
types of bacteria identified were monococcus, monobacillus and coccobacillus. The technique
used is not very effective because it is labor intensive and time consuming carrying since it took
five days to observe the bacteria under a microscope. As well, the test results can’t be guaranteed
to be completely accurate since bacteria have ability to mutate constantly to adapt to varying
environments thus they occasionally express genes that change a key biochemical and
morphological characteristics rendering this technique inadequate especially in clinical settings.
RECOMMENDATIONS
During sterilization, a microwave oven or hot air oven should be used since some bacterial
strains are hyper thermophilic and may survive at temperatures above that of the autoclave used
and therefore be spread to organisms and some microbes on apparatus may survive and interfere
with results of bacterial colonies.
In the process of inoculation, the source of the inoculum is picked should be selected with much
care so as to prevent picking highly infectious bacterial strains which may turn out hazardous
once cultured in sub-standardized laboratories.

8. Ssusume Patrick - DSTVE Makerere University
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2. Glenn and Suzzan Toole ( 1999 ): Understanding Biology,4th
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2022
4. S. Kumar (2012): Textbook of microbiology, Jaypee brothers medical publishers (P)
LTD. 1st
edition pg 20, pg 35-44, pg 57-67, pg 70-73.
5. Weaver, Hedrick (1997): Genetic WMC Broown Publishers, MC Graw-Hill Companies
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