PART 1: BASIC LABORATORY TECHNIQUES FOR ISOLATION, CULTIVATION AND

CULTURAL CHARACTERIZATION OF MICROORGANISM

BMY3201

BASIC MICROBIOLOGY TECHNIQUES

Num. Name Matric number

1. Muhammad Zulfaiz Bin Zuikarnain 201298
2. Aida Zahirah Binti Ahmad Fauzi 200426
3. Mumtaz Binti Ibrahim 201047
4. Huang Yifei 201596
5. Yong Hui Yi 202362
6. Nur Nasuha Najwa Binti Mohd Jais 200279

FACULTY OF BIOTECHNOLOGY AND BIOMELCULAR SCIENCES

DEPARTMENT OF MICROBIOLOGY

UNIVERSITI PUTRA MALAYSIA

OCTOBER 2019
1.0 Abstract

Microorganisms are found everywhere. They usually exist in a mixed culture in order to do
a research about one type of culture, pure culture is needed. Through this experiment,
subculturing method is conducted. This method need to be done using aseptic technique. In
Experiment 1 the technique for aseptic removal and transfer of microorganisms for
subculturing is carried out by using aseptic technique. In the end of experiment red
pigmentation should be seen in solid media while the broth turns to cloudy as the
microorganisms are present. In experiment two, both of the isolation technique which is streak-
plate technique and spread-plate technique are carried out to isolate discrete colonies from a
mixed culture like a mixture of Serratia marcescens and Staphylococcus aureus and a mixture
of Staphylococcus aureus and Escherichia coli as well as environmental specimen. The
purpose of spread plating and streak plating is to isolate individual bacterial cells (colony-
forming units) on a nutrient medium. Both procedures which is spread plating and streak
plating require understanding of the aseptic technique. The isolated culture can also be
identified based on their form, elevation, pigmentation and size. These discrete colonies is
observed after been cultured in difference media. In experiment three, Microorganisms exhibit
differences in the macroscopic appearance of their growth called cultural characteristics, used
as a basis for separating ,microorganisms into taxonomic groups. The cultural characteristics
are determined by culturing the organisms on nutrient agar slants and plates,in nutrient
broth,and in nutrient gelatin.

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2.0 Introduction

Microorganisms are present in our surrounding. However, they usually exist in mixed
populations. To study the microorganisms pure culture is produced. This helps to identify the
characteristics, metabolism, shape and nutrient of a type of microorganisms. In this
experiment we knew that to transfer microorganisms subculturing method is used. This
technique is important as it is would be routinely used in experiments ahead. In experiment 1
pure culture is used. Pure culture is a culture in which only one types of microorganisms exist.
In this experiment the aseptic technique is carried out. This technique is used to ensure the
experiment is conducted in sterilized environment which would avoided the culture from being
contaminated. The instrument such as needle, loop, test tubes used are flamed to keep sterile
before and after the experiment conducted. This technique would not be compromised as the
results would be affected. Proper sterilization would minimize the risk of infection towards the
culture. Finally, the method of holding the test tube during experiment is also essential in this
experiment. The tubes must be held properly according to the technique. Then, the method of
removing and replacing the cap are also essential which need to be done in sterile method so
that the infections is minimized.

Pure culture is laboratory culture that contain only a single unadulterated species of
cells.(Cappuccino & Welsh) A pure culture actually derived from a mixed culture by
transferring a small sample into new, sterile growth medium in such a manner as to disperse
the individual cells across the medium surface or by thinning the sample manyfold before
inoculating the new medium.(The Editors of Encyclopedia Britannica, July 1998) The streak-
plate and spread-plate inoculation is performed to separate the cells of a mixed culture so that
discrete colonies can be isolated to become pure cultures. The isolation of pure cultures is the
most important diagnostic tool used in industrial, agricultural production, a clinical or research
laboratory. Both techniques of isolation of pure cultures are used routinely in preparing and
maintaining stock cultures as well as in microbiological test procedures. The streak-plate
method is used most commonly to isolate pure cultures of bacteria. A loopful of mixed culture
is placed on the tip of an inoculation loop or needle and is streaked across the surface of the
agar medium. The successive streaks will dilute the inoculum sufficiently and the micro-
organisms are separated from each other. The spread plate method is a technique to plate a
liquid sample containing bacteria so that the bacteria are easy to count and isolate. During
inoculation, the culture is spread over with L-shaped bent glass rod while the Petri dish is spun
on a “Lazy Susan” turntable. (Cappuccino & Welsh) A successful spread plate will have a
countable number of isolated bacterial colonies evenly distributed on the plate. Once discrete,
the well-separated colonies can be picked up with a sterile needle and streaking it into
separate nutrient agar slants.

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 Cultural Characteristic of Microorganism is a way of studying the overall characteristic and
morphology of the microorganism itself. It is a very important study in the Microbiology fields.
This is because, different type of microorganism have different shapes, characteristic, colour
and appearance. It is almost like a first step of identification of the microorganism or the culture.
Indeed, it is also serves as a basis or the taxonomic identification of the microorganism. In the
experiment, the microorganism culture has been provided. An aseptic techniques must be
followed carefully in order to have a correct result without any contamination. All the material
and apparatus must be handled accordingly whilst doing the experiment to ensure the result
taken are also correct. For the nutrient agar slants experiment, the result taken includes the
abundance of growth, pigmentation, the form in which the appearance and also the
consistency. For the nutrient agar plates, the size, pigmentation, form, margin and elevation
are observed and recorded. For nutrient broth cultures, only the appearance of the growth are
recorded. Last but not least, for the nutrient gelatine, the liquefaction of the medium are
observe as liquidity or solidify and also the pattern of the liquefaction.

3.0 Material and Method

Material experiment one

Equipment used are Bunsen burner, transfer loop, transfer needle, marker pen and
test tube. Cultures used are nutrient broth and nutrient agar slant cultures of Serratia
marcescens. Media used are three nutrient broth tubes, three nutrient agar slants and three
nutrient agar deep tubes.

Methodology experiment one

The tube to been inoculated is labelled with the name of culture, media and date. Then
the tube and the culture was held in the palm of hand and secure it with thumb. The tubes was
separated to form a V. Using aseptic technique, the following transfer is carried out;

a) Slant to broth transfer

b) Broth to slant transfer

c) Slant to agar deep transfer

Aseptic technique been used to ensure the experiment conducted in sterilized

environment. Firstly, the needle or loop need to be sterilized by flaming it 45° towards Bunsen
burner until it became red hot. Next, the needle or loop is cooled down for a few seconds to
avoid from killing the culture. Then, the tubes held on the hand is uncapped using the hands

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that used to hold the needle. The cap was held, as it would compromise the aseptic technique
if it is put on the laboratory bench. After that, the necks of tubes are flamed. A loop or needle
used to obtain sample from the medium culture using particular method. For a slant to agar
deep tube transfer, needle is used by using stab inoculation method which the needle is
inserted until the bottom of the tubes in a straight line. For broth to slant transfer, loop been
used which the loop is lightly drew over the slant surface with zigzag motion. The slant to broth
transfer the loop is also used but the loop is shaked to dislodge the microorganisms in the
broth. Lastly, the tubes is reflamed before it is recapped as aseptic procedure would not be
compromised. The loop or needle is also reflamed to destroy any microorganisms left. All the
cultures were incubated at 25℃-27℃ for 24 hours.

Material experiment two

There is two parts in experiment 2 which is Part A and Part B. Experiment for Part A is
about isolation of discrete colonies from a mixed culture. For Part A, the cultures used are 24-
to 48-hour nutrient broth cultures of a mixture of Serratia marcescens and Staphylococcus
aureus and a mixture of Staphylococcus aureus and Escherichia coli. For the environmental
sample, we took the window in the laboratory. The media used in Part A are four Trypticase™
soy agar plates per group for each inoculation technique to be performed. The equipment
used are Bunsen burner, inoculating loop, glassware-marking pencil, culture tubes containing
1 ml of sterile water, test tube rack and sterile cotton swabs. Experiment for Part B is about
isolation of pure cultures from a Spread-Plate or Streak-Plate preparation. For Part B, the
cultures used are mixed-cultures, nutrient agar streak-plate and spread-plate preparations of
Serratia marcescens and Staphylococcus aureus, Escherichia coli and Staphylococcus
aureus and the environment specimen for Part A. The media used in Part B are three
Trypticase™ soy agar slants per group and for the equipment; we used Bunsen burner,
inoculating needle and glassware marking pencil.

Methodology experiment two

Part A: Isolation of Discrete Colonies from a Mixed Culture

For the streaking for isolation by the quadrant method, the back of petri dish is labelled
with our group name, date of experiment and the name of bacterium we used which are “S.m
+ S.a” and “S.a + E.c”. Mark the area with numbers on bottom of plate to divide the circle into
four quadrants. An inoculating loop held in at an angle through the flame of a Bunsen burner
until the entire length of the wire becomes glowing red from the heat. The wire is cooled by
touching it at periphery of plate. The neck of the Bijou bottle is flamed before obtaining the
inoculum. The sterile loop is inserted and a loopful of culture is obtained. The mouth of tube

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is flamed and recapped. The loopful of culture is dragged rapidly several times across the
surface of Area 1. The loop is reflamed and cooled. The Petri dish is turned 90°. The loop is
touched to a corner of the culture in Area 1 and dragged several times across the agar in Area
2. The loop should never enter Area 1 again. The loop is reflamed and cooled again and the
petri dish is turned 90°. Then, Area 3 streaked in the same manner as Area 2. Without flaming
the loop, the dish is turned 90° again. The culture dragged from a corner of Area 3 across
Area 4 using a wider streak. All plates is incubated in an inverted position from 24 to 48 hours
at 25°C .

For the preparation of environmental specimen, two petri dish is labelled properly with
our group name, date of experiment and the organism we used. A sterile cotton swab is
damped with sterile water and the excess water is wrung out by pressing the wet swab against
the walls of the tubes. An environmental specimen is obtained from the window of laboratory
with moistened cotton swab. The contaminated swab is placed back into the tube of distilled
water and mixed gently. The tube is let stood for 5 minutes. Then, a few dots are made on
both petri dish by using the swab.

A four-way streak-plate inoculation is carried out. A inoculating loop is flamed and

cooled by touching an unused part of the agar surface close to the periphery of the plate. The
loop was dragged rapidly several times across the surface of Area 1. The loop is reflamed and
cooled. The Petri dish is turned 90°. The loop is touched to a corner of the culture in Area 1
and dragged several times across the agar in Area 2. The loop should never enter Area 1
again. The loop is reflamed and cooled again and the petri dish is turned 90°. Then, Area 3
was streaked in the same manner as Area 2. Without flaming the loop, the dish is turned 90°
again. The culture was dragged from a corner of Area 3 across Area 4 using a wider streak.
All plates were placed in the incubation racks.

A spread-plate technique was carried out. The bent glass rod was immersed with 95%
alcohol in the beaker. The glass rod was removed from the beaker and it is passed through
the Bunsen burner flame with the bent portion of the rod pointing downward to prevent the
burning alcohol from running down the arm. The alcohol allowed to burn off the rod completely.
The rod was cooled for 10 to 15 seconds. The petri dish is turned and lightly touched the sterile
bent rod to the surface of the agar and moved it back and forth. This will spread the culture
over the agar surface. Plates is placed in the incubation racks. The racks will be placed in the
incubators.

Part B : Isolation of Pure Cultures from a Spread-Plate or Streak-Plate Preparation

An inoculating loop were held in at an angle through the flame of a Bunsen burner until
the wire becomes red hot. The wire was allowed to cool. The colony which is far from each

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other is touched. A small amount of colony was removed. A single-line streak was made which
is starting at the base of agar slant to the top. The inoculating loop is flamed. The slants ware
incubated at 37°C for 18 to 24 hours. All the slants were observed. The type of growth and
pigmentation was recorded. The name of organism was recorded.

Material experiment three

For culture substances, the material used were, Twenty-four-hour of nutrient broth
culture of Pseudomonas aeriginosa, Bacillus cereus, Micrococcus luteus and Escherichia coli.
Seventy-two- to 96-hour soy broth culture of Mycobacterium smegmatis.. Media used were,
nutrient agar slants, nutrient agar plates, nutrient broth tubes, and nutrient gelatine tubes.
Lastly the Bunsen burner, inoculating loop and needle, marker pen were used as the
equipment.

Methodology experiment three

In nutrient agar slant,a single line streak made of each of the cultures provided using
a sterile needle,started at the but and the needle drew up the center of the slanted agar surface.
For nutrient agar plates,a streak plate inoculation of each of the cultures prepared for the
isolation of discrete colonies. Moreover, in nutrient broth cultures,each organism inoculated
into a tube of nutrient broth using a sterile loop. The loop was shook for a few times to dislodge
the inoculum. A stab inoculation for each of cultures in nutrient gelatin prepared using a sterile
needle. All cultures incubated at 37 degree Celsius for 24 to 48 hours.

The gelatin cultures placed in a refrigerator for 30 minutes in a beaker of crushed ice for a few
minutes before observations. The gelatin culture was the last to be observed. Cultural
characteristics of bacteria shown in Figure 3.1 on page 42 and description in introductory
section of Experiment 3 referred to while following observations made. Each of nutrient agar
slant cultures observed for the amount,pigmentation, form and consistency of growth. A
single,well-isolated colony on each of the nutrient agar plate cultures observed and its size,
elevation , margin, form and pigmentation observed. Observations recorded in the chart
provided in the Lab Report. Each of the nutrient broth cultures observed for the appearance
of growth

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4.0 Result
Experiment one:

Figure 1:observation of culture S.marcescens

Figure 2:observations of culture B

Figure 3: observations of culture A

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 Growth Orange Red
Pigmentation

Culture A Nutrient Broth + -

Nutrient Agar Slants + +

Nutrient Agar Deep + +

Culture B Nutrient Broth - -

Nutrient Agar Slants - -

Nutrient Agar Deep - -

CULTURE Nutrient Broth + -

Serratia
Nutrient Agar Slants + +
Marcescens
Nutrient Agar Deep + +

Table 1 shows the result of the culture A, B and Serratia Marcescens

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Experiment two:

Part A: Isolation of discrete colonies from a mixed culture

STREAK-PLATE TECHNIQUE

S. marcescens and S. aureus S. aureus and E. coli

Draw the
colonies that
appear on
each agar
plate

Colony Isolate 1 Isolate 2 Isolate 3 Isolate4

description

Form Circular Circular Circular Circular

Elevation Raised Raised Raised Raised

pigmentation Yellowish-white White Yellowish-white Transparent

Size Small Small Small Pinpoint

Table 2.1 shows the pattern of growth on agar in petri dishes of the mixture of
Staphylococcus aureus with Serratia marcescens and mixture of Staphylococcus aureus with
Escherichia coli through streak-plate inoculation and also the colony description including the
forms , elevation, pigmentation and size.

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 ENVIRONMENTAL SPECIMEN

Spread-plate Technique Streak-plate Technique

Draw the
colonies that
appear on
each agar
plate

Colony Isolate 1 Isolate 2

description

form - Circular

elevation - Raised

pigmentation - White

Size - Pinpoint

Table 2.2 shows the pattern of on agar of petri dish of microorganisms in environmental
specimen which uses spread-plate technique and streak-plate technique inoculation and also
the colony description including the form, elevation, pigmentation and size.

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PART B：Isolation of pure cultures from a spread-plate or streak-plate preparation

Distribution
of growth on
the slant
surface

Type of Echinulate Echinulate -

growth

Pigmentation Yellowish-white Transparent -

Name of Staphylococcus aureus Escherichia coli -

organism

Table 2.3 shows the distribution of growth of microorganisms which isolated from pure
culture from a spread-plate or streak-plate and also the type of growth, pigmentation and name
of organism.

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 Experiment three:
NUTRIENT AGAR SLANT CULTURES

M. luteus P. aeruginosa M. smegmatis E. coli B. cereus

The
distribution
of
growth
on
the
slant
surface

Amount of slight large moderate moderate slight

growth

Pigmentation white green white white white

Form effuse echinulate beaded echinoculate echinoculate

Consistency dry dry buttery Shiny buttery uttery

Table 3.1 represents the observation of distribution of growth of slant surface

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 Nutrient Agar Plates

M. luteus P. aeruginosa M. smegmatis

The distribution
of colonies

Size pinpoint large small

Elevation flat flat umbonate

Margin entire entire undulate

Form circular irregular circular

Pigmentation white green white

Table 3.2.1 shows the observation of distribution of colonies on nutrient agar plates

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 The distribution Nutrient Agar Plates
of colonies
E. coli B.cereus

Size moderate large

Elevation raised umbonate

Margin undulate undulate

Form circular irregular

Pigmentation white white

Table 3.2.2 shows the observation of distribution of colonies on nutrient agar plates

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 Nutrient Broth Cultures

P. aerugi
M. luteus M. smegmatis E. coli B. cereus
nosa

Distribution
of the
growth

Appearance
Uniform Pellicle Flocculent Uniform Uniform
of growth

Table 3.3 shows the result of Nutrient broth culture

Nutrient Gelatin Cultures

M. luteus P. aeruginosa M. smegmatis E. coli B. cereus

Liquefaction
patterns

Liquefaction - + - - +

Type of
No change Stratiform No change No change Crateriform
liquefaction

Table 3.4 shows the result of Nutrient gelatin.

5.0 Discussion

In the experiment 1, the growth of microorganisms can be observed through the

nutrient broth, nutrient agar slant and nutrient agar deep in culture A. Orange-red pigmentation
was observed on the surface of nutrient agar slant and nutrient agar deep while nutrient broth
looked cloudy compared to culture B that acted as controller in the experiment. The results
shown that there were positive growth of S. marcescens when the inoculum was obtained
from all the medium.

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 In culture S. marcescens, the growth of microorganisms was obtained from all the
medium. Orange-red pigmentation were obtained on the surface of nutrient agar slant as
slanted surface area maximizes the surface area for microorganisms to grow as they get more
oxygen. Meanwhile, the gas exchange between nutrient agar deep was impeded by the height
of the agar in which can be observed on the top of the agar as the microorganisms was
concentrated at one place so it became redder. Thus, it shown that there were positive growth
of S. marcescens. Meanwhile, nutrient broth was obtained cloudy in colour compared to
culture B which was the controller of the experiment.

In culture B, that acted as the controller in the experiment, the inoculum could not
obtain the present of S. marcescens. Thus, there were no orange-red pigmentation found on
the surface of nutrient agar slant and nutrient agar deep. Furthermore, nutrient broth was
obtained clear in colour compared to culture A.

In the experiment, flamed the inoculating instrument prior to and after each inoculation
was essential during subculturing because it helps to maintain sterility and it helps to obtain
an accurate result. To achieve sterility, it is mandatory to sterile equipment and employed
aseptic techniques when handling bacterial cultures. As microorganisms always present in the
air, on the laboratory surfaces, benches and equipment thus it was quite crucial to sterilize
inoculating instruments because if the loop was not flamed between each step, the bacteria
from the previous step reintroduced and contaminated the culture. Furthermore, held the test
tube caps in the hand between fingers to avoid any contaminated to the test tube caps as
there were bacteria and contamination on our hands. It helps reduces the opportunity for
students been exposed to potential pathogens. Besides, cooling the inoculating instrument
prior to obtain the inoculum was one of the essential steps because excessive heat can kill
the bacteria that need to be obtained and it may lead to inaccurate result of the experiment.
Thus, cooling the inoculating instrument was the crucial step in the experiment to see the
distribution growth of bacteria. Therefore, flaming the neck of the tubes immediately after
uncapping and before recapping helps to remove any dust or possible contaminants such as
any microbes from spreading in the nutrient base.

Ambient microorganisms are microorganisms that present in the surrounding

environment and they should not be present during subculturing as they can contaminated the
cultures which caused inaccurate in results. A straight inoculating needle used to inoculate an
agar deep tube to get to the bottom of the tube.

Lack of orange-red pigmentation in some of the growth in agar slant does not mean that
there is contamination. It could just mean that the inoculate of bacteria was failed and bacteria
did not produced pigment due to certain condition such as temperature. To determine the

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contamination in culture, the gram-stained preparation of both colonies suspected and a
pigmented colony should be made.

In experiment two, streak-plate and spread-plate method performed to separate

the cells of a mixed culture so that discrete colonies can be isolated. Streak-plate method is
essentially a dilution technique that involves spreading a loopful of culture over the surface of
an agar plate. (Cappucino & Welsh) The dilution or isolation by streaking method was first
developed by Loeffler and Gaffky in Koch's laboratory, which involves the dilution of bacteria
by systematically streaking them over the exterior of the agar in a petri dish to obtain isolated
colonies in which will then grow into quantity of cells, or isolated colonies. (Wikipedia) Spread
plate technique is the method of isolation and enumeration of microorganisms in a mixed
culture and distributing it evenly by using L-shaped bent glass rod.

In part A experiment, a mixture of Serratia marcescens and Staphylococcus aureus and

a mixture of Staphylococcus aureus and Escherichia coli are used. Another culture used in
streak-plate technique is environmental culture. Streak-plate technique has been used to
isolate discrete colonies. For spread-plate technique, only environmental specimen is used.
As precaution steps, the aseptic technique should be performed during transfer the mixed
culture to avoid any contamination of cultures. Besides, using a sterilized loop is also very
important to make sure that there is a decrease in bacteria leading to individual colonies.
Sometimes, the observation of a streak-plate shows more colonies in quadrant 4 than
quadrant 3. This may due to the incorrect streaking technique or the loop was not sterilized
properly.

From the observation, the isolate 1 is circular, raised, small and has yellowish-white
colour while the isolate 2 is circular, raised, small and has white colour. The pigmentation of
Serratia marcescens should show red which is usually seen instead of white Serratia
marcescens do not show red pigment as it only produce red pigment, known as prodigiosin in
room temperature. Therefore, Serratia marcescens do not show red maybe because the
bacteria was incubated in high temperature. For the mixture of Staphylococcus aureus and
Escherichia coli, the isolate 3 is circular, raised, small and has yellowish-white pigment while
the isolate 4 is circular, raised, pinpoint and has white pigment. Since both isolate 1 and isolate
3 has yellowish-white pigment, we can conclude that both isolate are Staphylococcus aureus.
Therefore, the isolate 2 is Escherichia coli because it has white pigment.

For the environmental specimen, both streak-plate technique and spread-plate

technique are used. The environmental specimen used is window in the laboratory. A bent
glass rod is placed into a beaker and added sufficient amount of 95% alcohol to cover the
lower, bent portion. 95% alcohol is used as glass has low heat conductivity. By using 95%

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alcohol, the glass can easily burned and the glass rod is sterised more efficiently. From the
observation, there is no discrete colonies can be found in the agar plate that use spread-plate
technique. This is because most probably we performed the wrong spreading technique.
Based on our observation for streak-plate technique, there is a few colonies which is circular,
raised, pinpoint and has white pigment formed on the plane.

In Part B experiment, the pure culture from spread-plate or streak-plate preparation in

part A is isolated. Our group has isolated the Staphylococcus aureus, Escherichia coli and
environmental specimen into nutrient agar slants. The one which have the same echinulate of
growing type with yellowish-white pigment is Staphylococcus aureus. The type of growth which
is also echinulate with yellowish-white pigment is Escherichia coli. For the environmental
specimen, there is nothing shown on the slant tube. We are unable to observe any
pigmentation and there is no growth on slant surface. This may due to the improper technique
we used when transfer the colony.

From experiment 3, the nutrient agar slant which contained Micrococcus luteus,slight
amount of growth with white pigmentation,and the form effuse and dry consistency could be
observed. The nutrient agar slant which contained Pseudomonas aeruginosa, large amount
of growth with green pigmentation,and the form echinulate and dry consistency could be
observed. Moreover, nutrient agar slant which contained Serratia marcescens, moderate
amount of growth with white pigmentation,and the form beaded and buttery consistency could
be observed. The nutrient agar slant which contained Escherichia coli had moderate amount
of growth, with white pigmentation, and the echinoculate and shiny buttery consistency could
be observed. In addition, the nutrient agar slant which contained Bacillus cereus, slight
amount of growth with white pigmentation, and the echinoculate form and buttery consistency
could be observed. On all nutrient agar slants cultures, positive growth was shown.

The nutrient agar plate streaked with Micrococcus luteus displayed colonies in pinpoint
size with flat elevation,entire margin , circular form and white pigmentation.On the surface of
the nutrient agar slant containing Pseudomonas aeruginosa,large size colonies could be
observed.Flat elevation,entire margin, irregular form and green pigmentation were displayed.
The nutrient agar plate streaked with Serratia marcescens displayed colonies in small size
with umbonate elevation, undulate margin , circular form and white pigmentation. Moreover,
the nutrient agar plate streaked with Escherichia coli displayed colonies in moderate size with
raised elevation,undulate margin , circular form and white pigmentation. On the surface of the
nutrient agar slant containing Bacillus cereus,large size colonies could be observed.

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Umbonate elevation, undulate margin, irregular form and white pigmentation were displayed.
On all nutrient agar, positive growth was shown.

Based on the result of the table 3.3, Micrococcus luteus shows a uniform growth
appearance. The culture inside the medium are in uniform fine turbidity whereas the bacterium
are all over the places inside the medium. For Pseudomonas aeriginosa, pellicle appearance
has been observed. This indicates that the bacteria are more likely to be concentrated at the
top of the medium whilst, least concentrated in all over the medium. Thirdly the Mycobacterium
smegmatis, flocculent appearance formed in the broth. This is due to the bacterium inside
aggregates dispersed throughout. For Escherichia coli and Bacillus cereus, uniform
appearance observed. Both bacterium culture spread throughout the nutrient broth evenly.

Following the result from the table 3.4, the result for some culture are vary from one to
another. For Micrococcus luteus, Escherichia coli and Mycobacterium smegmatis, there was
no change in term of the solidify of the nutrient gelatine culture and no sign of gelatinase were
observed. For Pseudomonas aeriginosa, stratiform appearance of liquefaction were observed.
Stratiform means a complete liquefaction of the upper half of the nutrient gelatine. It happens
due to the high rate of bacterium binary fusion in which the bacterium doubled quickly.
Meanwhile, for Bacillus cereus, crateriform appearance can be observed in which the liquefied
surface area is saucer-shaped.

6.0 Conclusion

In conclusion, microorganisms were transferred from one medium to another by subculturing

in the experiment 1. For example, the inoculating loop was flamed in order to sterilize any
remaining organisms from contamination. Besides, the test tube closure must be keep in the
hand point away from the palm to avoid any contamination. Moreover, the positive growth of
microorganisms can be seen in culture A and S. marcescens and orange-red pigmentation
can be observed on nutrient agar slant, nutrient agar deep and nutrient broth. While there was
negative growth of microorganisms in culture B as it acted as controller and no orange-red
pigmentation presence. For experiment two, Streak-plate technique and spread-plate
technique are important to separate the cells of a mixed culture so that discrete colonies can
be isolated in scientific studies. Samples can then be taken from the resulting isolated colonies
and a microbiological culture can be grown on a new plate so that the organism can be
identified, studied or tested. Both methods are important diagnostic tool used in a clinical or
research laboratory as when the bacteria is streaked and isolated, the causative agent of a
bacterial disease can be identified. Lastly for experiment three, On all the nutrient agar
slants,nutrient agar plates,nutrient broths and nutrient gelatins, positive growth could be

19
observed. Different ways were used to identify the microorganisms such as observing the size
and form. Nutrient gelatin was used to observe gelatinase positive organisms and gelatinase
negative organisms. Morever, the stock culture of organism using isolates from mixed cultures
prepared on agar streak plates or agar spread plate was successfully prepared.

7.0 References

1. Cappucino.J.G.,and Welsh,C.(2018).Microbiology: A Laboratory Manual Eleventh

Edition. ESSEX CM20 2JE,England: Pearson

2. The Editors of Encyclopeadia Britannica, July 1998 from

https://www.britannica.com/science/pure-culture
3. https://en.wikipedia.org/wiki/Streaking_(microbiology)
4. KAZUE TAKEUCHI AND JOSEPH F. FRANK*. (1999). Penetration of Escherichia coli
O157:H7 into Lettuce Tissues as Affected by Inoculum Size and Temperature and the
Effect of. Journal of food protection, 434-440.
5. MARCIA TORO, ROSARIO AZCO´ N, AND JOSE-MIGUEL BAREA*. (1997).
Improvement of Arbuscular Mycorrhiza Development by Inoculation of Soil with
Phosphate-Solubilizing Rhizobacteria To Improve Rock Phosphate Bioavailability (32P)
and Nutrient Cycling. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 201-400.

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