UNIVERSITI SAINS MALAYSIA

PUSAT PENGAJIAN SAINS KAJIHAYAT

General Microbiology
Course code: BOI 207/3

Name: Yeap Qi Chen

Matric No.: 130255
Experiment: 4 - Pure Culture Technique
Date of experiment: 13 April 2017
Group: 8
Introduction
A microbiological culture is a method of multiplying microbial organisms by
letting them to reproduce in designed culture media under controlled laboratory
conditions. Microbial cultures are used to determine the type of organism, its
abundance in the sample being tested, or both. Microbiological cultures are
fundamental diagnostic methods used extensively as a research tool in molecular
biology. It is often essential to isolate a pure culture of microorganisms.
Pure culture is a laboratory culture containing a single species of organism. A
pure culture is usually derived from a mixed culture (one containing many species) 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
manifold before inoculating the new medium. Both methods separate the individual
cells so that each will form a discrete colony when they go through repeated
multiplication. There are three methods commonly used to derive a pure culture which
are spread plate, pour plate and streak plate. These methods dilute a heavy population
of bacteria across an agar surface.
Streak plate method is designed to isolate pure cultures of bacteria, or
colonies, from mixed populations by simple mechanical separation. A mixture of cells
is spread over the surface of a semi solid, agar based nutrient medium in a Petri dish
by streak plate method such that fewer and fewer bacterial cells are deposited at
widely separated points on the surface of the medium and develop into colonies after
incubation. The streak method for isolating single colonies from a mixture of cells is
accomplished with a metal sterile inoculating loop. The process is called “picking
colonies” when it is done from an agar plate with isolated colonies and is transferred
to a new agar plate using a sterile loop. The inoculating loop is used to streak over an
agar surface. Many organisms are deposited on the initial region of the streak which
resulting in confluent growth and thus lesser microorganisms are deposited as the
steaking progresses. The streaking process will dilute out the sample that was placed
in the initial region of the agar surface. There are two common used streak patterns
which are a three sector “T streak” and a four quadrant streak methods.
Pour plate method is used to count the number of microorganisms in a mixed
sample, which is added to a molten agar medium prior to its solidification. The
process results in colonies uniformly distributed throughout the solid medium when
the appropriate sample dilution is plated. This technique is used to perform viable
plate counts, in which the total number of colony forming units within the agar and on
surface of the agar on a single plate is enumerated. The main principle of this method
is to dilute the inoculum in successive tubes containing liquefied agar medium to
permit a thorough distribution of bacterial cells within the medium. The mixed culture
of bacteria is diluted directly in tubes containing melted agar medium maintained in
the liquid state a temperature of 50 degree Celsius (agar solidifies below 42 degree
Celsius). The bacteria and the melted medium are mixed well and then the contents of
each tube are poured into separate Petri plates to solidify and then incubated. After
incubation, isolating colonies forming units within the agar and on surface of the agar
on a single plate. These isolated colonies are then transferred to another Petri plate to
ensure purity by using inoculating loop and streak plate method.
Objective
 To understand the principles of pure culture techniques
 To learn how to obtain a pure culture using streak plate and pour plate
methods

Materials and Apparatus

Streak plate
 Mixed culture of Serratia marcescens and Escherichia coli
 3 nutrient agar plates
 Inoculating loop

Pour plate
 Mixed culture of Serratia marcescens and Escherichia coli
 20 ml molten nutrient agar (3 plates)
 3 empty sterile Petri dishes
 Inoculating loop
 2 nutrient agar plates

Experimental procedure
Streak plate method
1. Lab bench is prepared by removing extraneous items and the surface of
the table is cleaned with disinfectant.
2. Agar plates are sterile thoroughly and labelled accordingly at the
bottom.
3. Using the T-method (as illustrated below), outline the sections on the
bottom of the agar plate.

4. The mixed
culture is
shook gently
to suspend the

microorganisms.
5. The inoculating loop is flamed until it is red-hot and cooled down
without letting the loop to contact any other things.
6. Cap of mixed culture is removed using the little finger and the mouth
of the tube is flamed.
7. The cap of the tube is prohibited to be placed down on the table.
8. The inoculating loop is inserted into the tube and a loopful of broth
with bacterial cultures is removed.
9. The mouth of the tube is flamed again before the cap is returned to the
tube and set upright in the tube rack.
10. A loopful of the bacterial broth is placed in the first section of agar
plate.

11. The inoculating loop is used to streak the bacterial broth on the agar
surface in the first section, then repeat step 5.
12. The sterile cooled inoculating loop is used again to streak on the agar
surface in the second section. Note that DO NOT take another loopful
of bacterial broth but just OVERLAP with the first section a few times.
Then repeat step 5.

13. Again, the sterile cooled inoculating loop is used to streak on the agar
surface in the third section. Note that DO NOT take another loopful of
bacterial broth but just OVERLAP with the second section only. Then
repeat step 5.
 14. The agar plates are incubated inverted at 37 degree Celsius for 1 day
and the agar plates will be observed to obtain results.
15. After a day of incubation, a single colony of each species of bacteria
from the incubated streak plate is picked up aseptically and is streaked
on the new agar nutrient plates to obtain a pure culture of each species
of bacteria.

Pour plate method

1. Three sterile Petri plates are labelled at the bottom respectively.
2. 3 tubes of nutrient agar are removed from 50 degree Celsius water bath.
3. Please be efficient when removing the nutrient agar to prevent the nutrient
agar from solidifying in the tube.
4. 3 loopful of the mixed cultures are obtained using sterile cooled inoculating
loop and transferred to the nutrient agar in tube 1.
5. Tube 1 is rolled vigorously between the palms of both hands to mix the
bacterial broth with nutrient agar. Please do not shake the tubes up and down
to prevent the medium from splashing out due to unsecure cap.
6. Sterile cooled inoculating loop is used to obtain and transfer 3 loopful of agar
nutrient from tube 1 to tube 2 immediately.
7. The mixed nutrient agar in tube 1 is poured into the plate which is labelled at
the bottom as “Plate 1”.
8. Tube 2 is rolled vigorously between the palms of both hands to mix the
bacterial broth with nutrient agar. Please do not shake the tubes up and down
to prevent the medium from splashing out due to unsecure cap.
9. Again, sterile cooled inoculating loop is used to obtain and transfer 3 loopful
of agar nutrient from tube 2 to tube 3 immediately.
10. The mixed nutrient agar in tube 2 is poured into the plate which is labelled at
the bottom as “Plate 2”.
11. Tube 3 is rolled vigorously between the palms of both hands to mix the
bacterial broth with nutrient agar. Please do not shake the tubes up and down
to prevent the medium from splashing out due to unsecure cap.
12. The mixed nutrient agar in tube 3 is poured into the plate which is labelled at
the bottom as “Plate 3”.
13. The media are left to solidify and the plates are incubated inverted at 30
degree Celsius for 1 day and the agar plates will be observed to obtain results.
14. After a day of incubation, a single colony of each species of bacteria from the
incubated plate 3 is picked up aseptically and is streaked on the new agar
nutrient plates to obtain a pure culture of each species of bacteria.
Results
A. Streak plate culture

Based on the picture above, we can observe that both e.coli and s.aureus are
successfully isolated in the agar plate. We can differentiate the bacteria based on the
colour of the colonies, which e.coli is pale in colour while s.aureus is yellowish.

B. Pour plate culture

a
According to the pictures above, we can observe that the number of colony of each
bacteria e.coli and s.aureus is reducing from plate 1 to plate 3. This situation happens
because of the serial dilution process. E.coli is able to grow within and on the surface
whereas s.aureus only grow on the surface.

C. Pure culture

Streak plate – e.coli Streak plate – s.aureus

 Pour plate – e.coli Pour plate – s. aureus

These are the pure cultures of e.coli and s.aureus that obtained from both streak
plate and pour plate methods. According to the pictures provided, we can
distinguish both bacteria by referring their morphological differences as each
bacterium have different colour, size, texture, and shape. In this experiment with
the pictures above, we can identify the bacteria based on their colour and size in
which e.coli appears to be larger and pale in colour whereas s.aureus appears to be
smaller and yellowish.

Discussion
Pure culture can be obtained by both streak and pour plate methods. In this
experiment, two species of bacteria which are e.coli and s.aureus are separated from
the mixed culture to be grown in pure culture.
Identification of the bacteria can be done by observing the morphology of the
bacteria colony which including shape, colour, size and surface of the bacterial
colony. According to the pictures provided, we can only identify the bacteria colonies
by colour, size and shape. E.coli is pale in colour, larger in size and circular in shape
while S.aureus is yellow in colour, smaller in size compared to E.coli and also circular
in shape. Idenfication of the bacterial colony is crucial as the studies on the
biochemical, antigenic and other characters of bacteria can be done only if the
bacteria colonies are available in pure form.
Both streak plate and pour plate method are used in this experiment. Streak
plate method is taking a loopful of bacteria from a slant and streak it to a melted
nutrient agar in different quadrant by overlapping the cultures from initial quadrant
for isolation of colonies only. Meanwhile, pour plate method is a serial dilution
process of isolation of pure culture from mixed culture by adding bacterial broth into
several tubes in sequence before plating into a melted nutrient agar in a Petri dish
respectively to count the number of the organism. Streak plate method allows the user
to get distinct separate colonies but there is higher probability of contamination prior
to isolation. However, pour plate method has the advantage of allowing the growth
and quantification of microorganisms but its disadvantage is picking subsurface
colonies would interrupt other colonies by digging out of the agar.
Aseptic technique is essential when conducting this experiment as to prevent
contamination on both streak plate and pour plate methods. For example, the
inoculating loop is sterile every time after transferring the bacteria. Nevertheless, the
transfer of bacterial broth from tube to tube in pour plate method must be done
immediately as the nutrient agar would solidify below the temperature of 42 degree
Celsius.

Observation and Questions

1. Show within the circle the distribution of the colonies in your streak plate,
did you get clear isolation of yellow S. aureus and white E. coli?

Staphylococcus
aureus

Escherichia coli

No. Both e.coli and s.aureus are not isolated clearly from each other although both
bacteria can be distinguished by observing their colours, sizes and shapes. E.coli
can be easily seen as the colonies are pale in colour and distinct circular in shape
while S.aureus can be observed as yellow in colour only.

2. Show the distribution if the colonies in the pour plate 2 or 3. Indicate

which colonies are growing on the medium and which are growing within
it.
The picture above shows the colonies in plate 3. As per seen, the bacterial colonies are
lesser from plate 1 to plate 3. E.coli colonies are growing both on and within the agar
medium while S.aureus colonies are growing only on the medium.

3. Compare your results from the streak plate and pour plate methods.
Which method achieved the best separation of species?\
Streak plate method is able to achieve better separation of species compared to
pour plate method because clear isolation of the bacterial colonies can be
obtained and observed by streak plate method.

4. Which species are able to grow both on the surface and within the
medium? In which location are the colonies larger? Why?
E.coli is able to grow both on the surface and within the medium. E.coli
colonies are larger on the surface because a loopful of the bacterial broth
which contains high amount of E.coli colonies was placed on the surface of
the medium at quadrant 1 before the streaking is started to overlap the
quadrants on the surface.

5. In regard to bacterial growth on the solid media, define the term

“colony”.
Colony is defiend as a visible cluster of microorganisms growing on the
surface of or within a solid medium, presumably cultured from a single cell.

6. What do the advantages of streak plate method compare to pour plate

method?
The advantages of streak plat method compared to pour plate method are clear
isolation of bacteria cultures into distinct separate colonies, no temperature
control needed for this method and the other colonies would not be
interrupted by digging out of the agar when picking up the subsurface
colonies. Using streak plate method, the original culture is diluted serially and
a small volume of the final dilution is spread on the surface of an agar plate. A
 dilution gradient is established across the face of the Petri dish as bacterial
cells are deposited on the agar surface at a distance from all others.

7. Before inoculating and pouring molten nutrient agar into a plate, why
must the agar first be cooled to 50℃?
Agar first be cooled to 50℃ in order to avoid colony mix-up with each other
spread throughout the plate surface because there is water evaporation from
media occurs and condenses on the lid of the Petri dish which may drop on the
colonies developed on the media surface. Agar is also kept in high temperature
in the initial and so cooling it to 50℃ is suitable for the transfer of the
bacterial colonies as the agar would solidify at the temperature below 42℃.

8. Provide two reasons why plates should be inverted during incubation.

The plates should be inverted during condensation because warm incubators
tend to attract more condensation and condensation in Petri dishes causes
bacterial samples to spread and potentially mix with each other. Inversion of
the plates allows water to drip down onto the lid, away from the agar rather
than onto bacteria. Besides that, the plates are inverted because the lid of the
Petri dish may open during handling when incubated in normal position and it
may cause contamination from air. So, the labelling is done at the bottom part
to read the labelling easier and avoid confusion.

Conclusion
Pure culture is the culture containing a single species of the organism. Pure
culture is essential for the identification of the bacteria as the studies on the
biochemical, antigenic and other characters of bacteria can be done only if the
bacteria colonies are available in pure form. Streak plate and pour plate method are
the techniques used to obtain pure culture. Aseptic technique should be conducted all
the time when conducting both streak and pour plate methods to avoid contaminations
of the bacterial colonies.
References
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