FAR 121/4

MICROBIOLOGY FOR PHARMACY

PRACTICAL REPORT 2:
A) EXAMINATION OF CULTURES FOR MORTALITY
B) CULTURE TECHNIQUE

Name

: Low Kah Jun

Matrix No

: 127920

No. IC

: 960526-06-5529

Date

: 5th October 2015

Lecturer

: Assoc. Prof. Dr. Hjh. Pazilah Ibrahim

I.

EXAMINATION OF CULTURES FOR MOTILITY

Objectives:
1. To identify and differentiate between true motility and Brownian motion.
2. To familiarize and perfect their aseptic techniques in
- handling aseptic transfers
- isolating a pure culture
Introduction:
A large number of microorganisms are more or less actively motile by flagella.
Flagella are long thread-like structures arranged in various ways on the bacterial cell. The
motility of an organism is often an important characteristic in its identification and/or
classification. In this exercise, students will be exposed to 2 methods of determining motility;
one is quick but requires a little skill with the microscope, the other is much easier to perform
but one has to allow 24-48 hours for incubation.
Materials:
Refer to FAR 121/4 MICROBIOLOGY FOR PHARMACY practical manual, page 5.
Method:
(A) Hanging Drop preparation
Refer to FAR 121/4 MICROBIOLOGY FOR PHARMACY practical manual, page 6.
(B) Soft agar stab
Refer to FAR 121/4 MICROBIOLOGY FOR PHARMACY practical manual, page 6.

Result:
(A) Hanging drop preparation

Observations

Explanation

Rod-shaped Pseudomonas aeruginosa

moves in a zigzagged-like pathway.
The bacteria move quickly in a
specific direction and exhibits true
motility.
The bacteria are motile due to the
presence of flagella.

motion of Pseudomonas aeruginosa

Pseudomonas aeruginosa
Magnification : 40 10

Irregular cluster of spherical shaped

Staphylococcus aureus either vibrates
in a fixed position or moves randomly
in any direction.
The bacteria move randomly in any
direction due to the collision with air
or water molecules.
The bacteria do not move in a specific
direction. It exhibits Brownian
movement.
The bacteria are non-motile due to the
lacking presence of flagella.

Staphylococcus aureus
Magnification: 40 10

(B) Soft agar stab

Observations

Explanation

Growth of Pseudomonas aerugina is

spread on the surface of the soft agar
and the single stab line (line of
inoculation) made by the straight wire.
Bacteria growth area is larger.
Pseudomonas aerugina is a motile
bacteria.

Pseudomonas aeruginosa

Growth of Staphylococcus aureus is

limited, just around the single stab
made by the straight wire on the soft
agar.
The stab line is very sharp and defines.
Bacteria growth area is smaller.
Staphylococcus aureus is a non-motile
bacterium.

Staphylococcus aureus

Discussion:
(A) Hanging Drop Method
Motility of microorganism is one of the methods that usually used to shows the presence
of the flagella in bacteria. For examples soft agar stab method, wet-mount technique and

hanging drop preparation and observation under the microscope. This method does not give
the details in amount and arrangement of the flagella but used to show the presence of
flagella. The motility can be divided into three types as below:
1. show true motility by moving in a definite direction.
2. brownian movement due to the collisions with surrounding particles.
3. non-motility by vibrating on the same spot.
After the hanging drop preparation, the slide are put on the stage of microscope,
Pseudomonas aeruginosa shows the movement in same and definite direction. This proved
that Pseudomonas aeruginosa possess flagella to aid in movement. Therefore, they show true
motility. By using the magnification of 400, motility of the Pseudomonas aeruginosa can be
observed.
For Staphylococcus aureus, it is moving in different indefinite directions and some
vibrating vigorously on the same spot. This called Brownian movement and is due to the
collision of the bacteria with the surrounding particles like suspending liquid and bacteria
which lead to the random motion. The bacteria also vibrate in the same rate. Therefore,
Staphylococcus aureus does not show true motility and are non-motile bacteria.

(B) Soft agar stab

This experiment is slow because requires 48 hours incubation to allow bacteria
growth. The pattern of growth of the colonies shows the motility of the bacteria. This method
is much easier to be carried out as it does not require skill with the microscope. In addition,
heat source of the light microscope may dry and kill bacteria in hanging drop method,
resulting in an incorrect result.
Pseudomonas aeruginosa grew around the stab line and on the surface of the agar.
Growth spread throughout the whole bottle rather than being focused along the line of
inoculation. This is because the presence of the flagella allows it to move further away from
the stab line and spread whole agar.
Staphylococcus aureus grew only along the line of inoculation within a limited area.
The growth pattern is sharp and defined. This showed that it is non-motile as it is unable to
move away from the stab line and spread around the agar medium due to the absence of
flagella.
Precautions:
1. Both the wire loop and the straight wire must be heated till red hot before and after using to
prevent contamination and both of them must be cooled down completely before taking the
bacteria from the agar plate or broth as the heat would kill the bacteria.

2. Aseptic techniques should be carried out during the experiment.

3. The light intensity of the microscope should be minimized to prevent the bacteria from
drying out and dying.
4. The culture broth should be gently shaken first before taking the bacteria sample in order to
get a constant mixture in the culture bottle.
5. During soft agar 'stab', the inoculating straight wire should be stabbed slowly and gently to
avoid the agar from breaking.
Conclusion:
Hanging drop method is faster in observe the result compare to soft agar stab which
needs incubation period of 24-48 hours. However, hanging drop method needs a higher skill
and meticulous procedure to ensure that observation can be done, while soft agar stab is
much easier to carry out and results can be observed obviously and easily. Both techniques
show clearly the motility of bacteria. Definite and consistent movement of bacteria is
considered to be true-motility while indefinite movement is called Brownian movement.
Therefore, Pseudomonas aeruginosa is a motile bacteria due to the presence of flagella.
Staphylococcus aureus is a non-motile bacteria.

II.

CULTURE TECHNIQUE

Introduction:
As most microorganisms are potentially pathogenic it is essential that they be handled
by procedures which avoid contamination of the microbiologist. Also since microorganisms

are so ubiquitous, it is necessary to avoid contamination of microbial culture by stray

microorganisms.

1) Inoculum transfer
a) Technique of transferring culture from one agar plate to another by streaking
Materials:
Refer to FAR 121/4 MICROBIOLOGY FOR PHARMACY practical manual, page 7.
Method:
Refer to FAR 121/4 MICROBIOLOGY FOR PHARMACY practical manual, page 7.
b) Technique of transferring culture from an agar plate to a bottle of nutrient broth
Materials:
Refer to FAR 121/4 MICROBIOLOGY FOR PHARMACY practical manual, page 8.
Method:
Refer to FAR 121/4 MICROBIOLOGY FOR PHARMACY practical manual, page 8.

Result:
1) Inoculum transfer
a) Technique of transferring culture from one agar plate to another by
streaking
Observations

Explanation

The colonies of Escherichia coli

are spread from the first streaking
and successfully separated into
colonies at the end of streaking.
The colonies are oval in shape
and milky yellowish in colour
Isolated colonies of Escherichia
coli are formed.
Streaking was successful.

Escherichia coli
( in agar plate)

b) Technique of transferring culture from an agar plate to a bottle of nutrient

broth
Observations

Explanation

Before incubation, the bottle of

nutrient broth is clear, transparent
and yellowish in colour.

After incubation, the bottle of

nutrient broth turned turbid due to
the rapid growth of Escherichia
coli because it contains the
metabolic
secretion
of
Escherichia coli.

Escherichia coli
After incubation
Discussion:
a) Technique of transferring culture from one agar plate to another by streaking.
Streaking is used to dilute the colonies of bacteria into small and isolated colonies and
must be done meticulously to ensure successful isolation. Streaking is done at least 4
times in order to obtain isolated cultures. Incubation has to be done at 37 for 24

to 48 hours. Different bacteria have different colours, textures, shapes and sizes. The
colonies of Escherichia coli are round in shape and milky yellowish in appearance.
b) Technique of transferring culture from an agar plate to a bottle of nutrient
broth.
The nutrient broth that was initially clear had turned turbid and milky showing
bacterial growth and metabolite production. This proved that the transfer was done
successfully.
Precautions:
1) Heat the wire loop and the straight wire until burning red and allowed to cool sufficiently
before and after handling the bacteria to avoid contamination and death of bacteria due to
heat.
2) Overlap every streaking with the following streaking except the first and the final streak.
Ensure last streak and the first streak must not be overlap to obtain isolated colonies.
3) As the agar is a fragile medium, so the streaking must be done carefully and gently.
4) The cover of the petri dish should be opened at a 45and is closed immediately after used
to minimize contamination by particles in the air.
5) The agar plate must be incubated in an inverted position. This is to prevent the vapour
formed on the cover of the petri dish by evaporation and condensation of water to drop
on the bacteria colonies and affect the growth.
6) The open side of the bottle must be flamed before and after handling bacteria.
7) All the transferring procedures should be done near and around the flame as the area is
assumed to be sterile.

Conclusion:
Isolation of colonies can be done by streaking method (agar plate) and incubation of agar
medium (bottle). The isolated colonies on agar plate and turbidity of nutrient broth can be
used to examine growth of bacteria. Escherichia coli were successfully transferred from one
agar plate to another and nutrient broth.

2) Isolating a pure culture from a mixed culture

Materials:
Refer to FAR 121/4 MICROBIOLOGY FOR PHARMACY practical manual, page 9.
Method:

Refer to FAR 121/4 MICROBIOLOGY FOR PHARMACY practical manual, page 9.

Result:
a) From a mixed culture plate to a partitioned agar plate
Observations

Explanation

Mixed culture of Bacillus

subtilis and Staphylococcus
aureus.
The colonies of Bacillus
subtilis are spherical in shape,
smaller and white (creamy) in
colour.
The colonies of
Staphylococcus aureus are
small, rounded, yellow and
milky.

Mixed culture of Bacillus subtilis and

Staphylococcus aureus colonies

b) From a mixed culture plate to nutrient broth

Observations

Explanation

Before incubation, the nutrient

broth was clear due to absence of
bacterial growth.

After incubation, the nutrient broth

turned cloudy and turbid due to
bacterial growth and metabolite of
Bacillus
subtilis
and
Staphylococcus aureus.

Transfer of bacteria was done

successfully.
After inoculation
Bacillus subtilis and Staphylococcus aureus

Discussion:
Streaking is a proper and suitable method to isolate bacteria colonies. A successful
streaking will yield colonies of bacteria from different species. A mixed culture of Bacillus
subtilis and Staphylococcus aureus was separated successfully using the streaking technique
on a nutrient agar plate. The isolated pure colonies of Bacillus subtilis are white (creamy) in
colour and circular. The isolated pure colonies of Staphylococcus aureus are milky yellow in
colour and rounded. The differences are obvious and visible by naked eyes.
Nevertheless, it is hard to isolate a pure culture from a mixed culture by using nutrient
broth because it is not easy to detect the contamination in broth culture. After transferring
mixed culture into a nutrient broth and incubated, the nutrient broth turned from clear yellow
to turbid and cloudy. The turbidity was caused by the metabolite products of the bacteria,
indicating growth of bacteria. The mixed culture was successfully transferred but no isolation
was observed. This shows that this method is not suitable for isolating colonies of bacteria
but is suitable for cultivating bacteria.

Precautions:
1. The wire loop must be sterilized by heating until burning red every time before
handling bacteria to avoid contamination of the wire loop.

2. Sufficient time must be given to cool the wire loop before handling bacteria to prevent
bacteria death by heat.
3. Every streaking must overlap the following streaking except the first and the final
streak. The last streak must not overlap the first streak to obtain isolated colonies.
4. Streaking must be done carefully and gently as the agar is a fragile medium.
5. The petri dish should be opened at a 45 degree angle and is closed immediately after
bacteria are obtained to avoid contamination by particles in the air.
6. The agar plate must be incubated in an inverted position to prevent the evaporation
and formation of vapour on the cover of the petri dish and condensation of water to
drop on the bacteria colonies and disturb the colonies.
7. The mouth of the bottle must be flamed before and after handling bacteria.
8. All the transferring procedures should be done near and around the flame as the area
is assumed to be sterile.
Conclusion:
Streaking bacteria from a mixed culture on an agar plate separately can be used to isolate
Bacillus subtilis and Staphylococcus aureus. The colonies of the two species can be
differentiate and obtained separately. Staphylococcus aureus are small, rounded, milky and
yellow; while Bacillus subtilis appears as spherical and white (creamy) in colour. Nutrient
broth can be used to growth bacteria, but it is not suitable for isolation of bacteria as all the
colonies will be mixed in the broth. This technique was successfully performed.

Results:
Part 1
a. Observe the streaked plate and make a sketch to show the distribution of growth.
Please refer to the observation of inoculum transfer in techniques of transferring culture from
one agar plate to another by streaking.
b. Were you able to streak properly so as to obtain isolated? Are the colonies similar to
those on the original plate?
Yes, I am able to streak properly so as to obtain isolated colonies. The isolated
colonies of Escherichia coli observed are similar to those on the original plate. The
colonies are round in shape and not transparent.
c. Observe the changes in the nutrient broth before and after inoculation. Why did the
inoculated broth turn turbid?
Before inoculation, the nutrient broth is clear and transparent and it becomes turbid
after inoculation. This is due to the growth of Escherichia coli in the nutrient broth
and the bacteria secrete waste products of metabolism during its growth.

d. Can you use a liquid medium to isolate a pure culture? Explain.

No, we cannot use a liquid medium to isolate a pure culture because it is not a suitable
method. We cannot determine whether the turbidity of the broth is caused by a pure
culture or the contamination of other microorganisms.

Part 2
a. Observe the streaked plate. Were you able to separate the 2 organisms successfully? If
not, why? Describe the morphology of the colonies produced: -colour, texture, shape
etc.
The mixed culture of Bacillus subtilis and Staphylococcus aureus are successfully
separated. The colonies of Bacillus subtilis are spherical in shape, smaller, smooth in
appearance and white (creamy) in colour, while the colonies of Staphylococcus
aureus are spherical in shape, bigger, rough in appearance and yellowish in colour.
b. How would you know if your culture has been contaminated?
We will know a culture is contaminated by the detection of different characteristics or
features from the true culture as different bacteria has different morphology, colour,
texture as well as shape.
c. Explain why you must incubate an inoculated plate in an inverted position.

Bacteria will carry out respiration which releases carbon dioxide and water under 37 .
Evaporation of water causes water droplets (vapour) formed at the cover. If the inoculated
plate was not placed in an inverted position, the water droplets will drip onto the surface of
agar. This will affect the formation of colonies of bacteria because the water will cause the
bacteria colonies to bind together as a medium for movement of bacteria, leading to an
inaccurate result.