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Isolation of Bacterial Cultures

Through Streak Plate Method

ISOLATION OF BACTERIA INTO PURE


CULTURES THROUGH STREAK PLATE METHOD

REYES, KIM RAFAELLE E.


Mapua Institute of Technology
Intramuros, Manila
Philippines
kimayna@yahoo.com

ABSTRACT
Streak plate technique is a common microbial technique used for the isolation into pure culture
of the organisms, mostly bacteria, from mixed population. Disinfection of working station was
done using standard Lysol disinfectant spray prior to the experiment. The inoculum is diluted by
streaking it across the surface of the agar plate. While streaking in successive areas of the plate,
the inoculum is diluted to the point where there is only one bacterial cell deposited every few
millimeters on the surface of the agar plate. Aseptic technique was observed throughout the
whole process. The students cooled the agar for two minutes. After that, the agar was then
wrapped in paper and plastic bag and is ready for incubation at 37C for about 48 hours.

INTRODUCTION
Once a culture medium has been prepared and made sterile to render it free of all life
forms, organisms can be inoculated and the culture can be incubated under conditions that will
support growth. In a laboratory, inoculation will typically be with a pure culture, a culture
containing only a single kind of microorganism. It is essential to prevent other organisms from
entering a pure culture. Such unwanted organisms, called contaminants, are ubiquitous and
microbiological techniques are designed to avoid contamination.
Streak plate technique is a common microbial technique used for the isolation into pure
culture of the organisms, mostly bacteria, from mixed population. The inoculum is streaked over
the agar surface in such a way that it thins out the bacteria. Some individual bacterial cells are
separated and well-spaced from each other. As the original sample is diluted by streaking it over
successive quadrants, the number of organisms decreases. Usually by the third or fourth quadrant
only a few organisms are transferred which will give discrete colony forming units (CFUs).
The sample/inoculum is diluted by streaking it across the surface of the agar plate. While
streaking in successive areas of the plate, the inoculum is diluted to the point where there is only
one bacterial cell deposited every few millimeters on the surface of the agar plate. When these
lone bacterial cells divide and give rise to thousands and thousands of new bacterial cells, an
isolated colony is formed. Pure cultures can be obtained by picking well isolated colonies and
re-streaking these on fresh agar plates.

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Isolation of Bacterial Cultures
Through Streak Plate Method

MATERIALS AND METHODS

Disinfection of workstations and sterilization of apparatuses. Before conducting any


experiment, the work station is disinfected using disinfecting spray (in this case Lysol is used).
Petri dishes and test tube which will be acting as a vessel to our bacterial colony are sterilized by
autoclaving. Test tubes are sealed with prepared cotton plugs and are foiled to avoid
contamination.

Sterilization of Inoculating Loop. Sterilize the inoculating loop in the alcohol lamp by putting
the loop into the flame until it is red hot. Allow it to cool.

Selecting a colony. Pick an isolated colony from the agar plate culture and spread it over the first
quadrant using close parallel streaks or Insert your loop into the tube/culture bottle and remove
some inoculum. Select the smallest colony for convenience.

Streaking the inoculating loop. Immediately streak the inoculating loop very gently over a
quarter of the plate using a back and forth motion as in Figure 1.

NOTE: Streak lightly so that you do not gouge the agar. Always flame the loop after you
streak each quadrant (90 turn).

Fig. 1

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Isolation of Bacterial Cultures
Through Streak Plate Method

Turn the petri dish into 90.Flame the loop again and allow it to cool. Going back to the edge
of area 1 that was streaked, extend the streaks into the second quarter of the plate as in Fig 2

Fig. 2
Turn the petri dish again into 90.Flame the loop again and allow it to cool. Going back to the
area that you just streaked, extend the streaks into the third quarter of the plate as in Fig. 3.

Fig. 3

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Isolation of Bacterial Cultures
Through Streak Plate Method

The last streak is the same as the previous ones. Flame the loop again and allow it to cool.
Turn the petri dish into 90 .Going back to the area that was just streaked (area 3), extend the
streaks into the center fourth of the plate.

Fig. 4

Storage and incubation. Invert the plate and incubate at 37C for 24 hr.

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Isolation of Bacterial Cultures
Through Streak Plate Method

RESULTS AND DISCUSSION

After 48 hours of incubation, the petri dishes were checked and the following results were
obtained.

(1)Previous mother culture: (2) Streak plate:

*A pure culture was obtained, as shown in the photo to the right (Streak plate).

Streak plate technique is used for the isolation into pure culture of the organisms (mostly
bacteria), from mixed population. To achieve this result the bacterial stock is streaked with an
inoculating loop onto solid media in a series of steps. As bacteria are streaked from one area of
the plate to the next, a dilution occurs. At the last streak, the number of cells on the loop should
be small. As this small number of cells is spread across the remaining area of the plate, the cells
are physically separated. Each isolated cell will divide. As the dividing cells accumulate in the
local region of the original isolated cell, a distinct mound of cells becomes visible.
The sample is diluted by streaking it across the surface of the agar plate. This was done to
spread the bacteria. While streaking in successive areas of the plate, the inoculum is diluted to
the point where there is only one bacterial cell deposited every few millimeters on the surface of
the agar plate. When these lone bacterial cells divide and give rise to thousands and thousands of
new bacterial cells, an isolated colony is formed. Pure cultures can be obtained by picking well
isolated colonies and re-streaking these on fresh agar plates. Common assumption is an isolated
colony of bacteria is the progeny of a single bacterial cell (i.e. colony is the clone). However,
this is not necessarily true. With species in which the cells form a characteristic grouping during
cell divisions, the colony forming unit may develop from a group of cells rather than form a
single cell.
Isolated colonies of different species will demonstrate distinct morphologies. The purity
of a culture can be determined by observation of the morphology of isolated colonies. In this
experiment the student identified the streak colony to be Circular in form and is elevated by a
few millimeters summarized in Fig 1.1

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Isolation of Bacterial Cultures
Through Streak Plate Method

Fig 1.1 (boxed)

A streak plate of a mixed culture will show colony types of each culture. To isolate pure
cultures of each culture found in a mixed culture, one distinct colony may be picked, restreaked
on a fresh culture plate and incubated. Observation of identical colonies on this second plate will
indicate a pure culture. Most microbiologists repeat streaking from an isolated colony two to
three times before they guarantee culture purity. For this particular experiment, the observable
colonies are unitary in shape and form which indicates that it is already a pure culture.
The use of the streak plate technique requires knowledge of rules of aseptic technique.
Whenever working with a culture, the microbiologist must proceed with care to ensure that the
culture is not contaminated during the manipulations, and with caution that no culture escapes
into the environment. In addition it is important that all culture vessels and laboratory media are
free from bacteria - sterile. Sterilization procedures include treatment with high temperature and
irradiation. Some culture media are filtered to remove all bacteria. Inoculating loops are exposed
to a flame for sterilization. For this experiment, the students used autoclaving for sterilization.
The petridish used were autoclaved at 121C for about three hours. In order for bacteria to grow,
the students incubated the plates at optimum temperature for bacterial growth which is 37C. The
incubation process should have been only for 24 hours but in this experiment, the students
incubated for 48 hours instead because of some technical circumstances.
The purpose of a streak plate is to produce colonies of a certain organism. These colonies
are formed by encouraging the growth of only one organism, which allows the separation of that
organism from a sample containing multiple organisms. The result of this experiment coincides
with the objective of harnessing a pure culture.

CONCLUSIONS
This study demonstrates the isolation of pure culture using the streak plate method.
Sterilization by autoclaving and Aeseptic technique used in the previous experiment were still
applied to avoid contamination. The experiment provided the desirable results.

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Isolation of Bacterial Cultures
Through Streak Plate Method

ACKNOWLEDGEMENT
This research was supported by Mr. Heherson Cabrera of the School of Che-Chm-BE-
MsE of the Mapua Institute of Technology.

REFERENCES
[1] Streak plate technique. (n.d.). Retrieved March 03, 2017, from http://www.biology-
online.org/dictionary/Streak_plate_technique
.
[2] What is the purpose of a streak plate? (n.d.). Retrieved March 03, 2017, from
https://www.reference.com/science/purpose-streak-plate-abcb49a8a1004716#

[3] Madigan, M. T., Martinko, J. M., & Parker, J. (1997). Biology of microorganisms: Brock.
New Jersey: Prentice Hall.