DIPLOMA IN PHARMACY COHORT 35

YEAR 3 SEMESTER 1

PAPM 321.2 PHARMACEUTICAL MICROBIOLOGY (P)

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LAB REPORT 2 : ISOLATION OF PURE CULTURE – STREAK METHOD

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PREPARED BY GROUP 4 :

 SAFIA ASYIFA BINTI ZAINAL ABIDIN (2041193056)

 ALYAA ATHIRA BINTI AMIR (2041193002)

 NURUL FATIHAH BINTI ARNUAR (2041193053)

 MAS FIRZA NATASHAH BINTI MAS ALINA (2041193068)

PREPARED TO :
SIR SYAFFUAN BIN AHMAD NAJIB
 INTRODUCTION

The streak plate technique is the most common method for isolating specific bacteria from
samples containing a mixture of microorganisms (Escherichia coli and Staphylococcus aureus)
in pure culture. This technique works by decreasing the number of organisms and their density.
Microbiologists can use it to identify and isolate specific bacterial colonies. A colony is a
visible bacterial grouping. In a single colony, all of the bacteria come from the same bacterial
cell. As a result, individual colonies are considered "pure". To make a pure culture of one
species of bacteria, the pure colony is transferred to another plate (nutrient agar plate).
Cultivation of pure cultures is absolutely necessary prior to performing biochemical tests to
identify suspected microorganisms in clinical laboratories.

OBJECTIVES

1) To practice aseptic technique.

2) To produce individual colonies on an agar plate, by using the streak plate technique.

MATERIALS

Inoculating Loop

Bunsen Burner

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 1 Nutrient Agar Plate (per group)

Test Tube Rack

Escherichia coli (Gram-negative)

Broth Culture / Nutrient Broth : mixed culture

containing both Staphylococcus aureus and
Escherichia coli Staphylococcus aureus (Gram-positive)

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 METHODS / PROCEDURE

THREE QUADRANTS STREAK FOUR QUADRANTS STREAK

1) Label and divide the bottom of a nutrient agar plate into 1) Label and divide the bottom of a nutrient agar plate into
three quadrants using a fine-tipped sharpie. Label it four quadrants using a fine-tipped sharpie. Label it with
with initials of the bacteria (mixed culture - E. coli & initials of the bacteria (mixed culture - E. coli &
Staphylococcus aureus), the date, and the type of Staphylococcus aureus), the date, and the type of
culture medium (nutrient agar). culture medium (nutrient agar).

2) Sterilize the inoculating loop by flaming it using the

2) Sterilize the inoculating loop by flaming it using the bunsen burner until it is red hot, and allow it to cool.
bunsen burner until it is red hot, and allow it to cool.

3) Remove the cap from the tube, sterilize by passing the

3) Remove the cap from the tube, sterilize by passing the
tube through the flame, and obtain a loop of bacteria
from the tube (nutrient broth) using the inoculating
loop. Then, pass the tube back through the flame before
recapping.
tube through the flame, and obtain a loop of bacteria
from the tube (nutrient broth) using the inoculating
loop. Then, pass the tube back through the flame before
recapping.

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4) Lift one side of the petri plate lid and immediately
streak about one quarter of the agar with a back and
forth motion using inoculating loop. Ensure to hold the
loop like a pencil and gently touch the surface of the
agar.

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 4) Lift one side of the petri plate lid and immediately
streak about one quarter of the agar with a back and
forth motion using inoculating loop. Ensure to hold the
loop like a pencil and gently touch the surface of the
agar.

5) Replace the lid and flame the loop to sterilize, and

allow it to cool. Then, turn the agar plate 90 degrees,
and by using sterilized inoculating loop, streak through
the edge of the first sector that just streaked, and extend
the streaks into the second sector (second quarter of the
agar) with a back and forth motion.

5) Replace the lid and flame the loop to sterilize, and

allow it to cool. Then, turn the agar plate 90 degrees,
and by using sterilized inoculating loop, streak through
the edge of the first sector that just streaked, and extend
the streaks into the second sector (second quarter of the
agar) with a back and forth motion.

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 6) Replace the lid and flame the loop to sterilize and allow
it to cool. Turn the agar plate 90 degrees, and by using
sterilized inoculating loop, streak through the edge of
the second sector that just streaked, and extend the
streaks into the third sector (third quarter of the agar)
with a back and forth motion.

6) Replace the lid and flame the loop to sterilize, and

allow it to cool. Turn the agar plate 90 degrees, and by
using sterilized inoculating loop, streak through the
edge of the second sector that just streaked, and extend
the streaks into the third sector (third quarter of the
agar) with a back and forth motion. Again, replace the 7) Replace the lid and flame the loop to sterilize and allow
lid and flame the loop. it to cool. Turn the plate 90 degrees, and by using
sterilized inoculating loop, streak through the edge of
the third sector that just streaked, and extend the
streaks into the fourth sector (fourth quarter of the agar)
with a back and forth motion. Again, replace the lid and
flame the loop.

8) Incubate the nutrient agar plate in an inverted position

at 35°C to 37°C for at least 24 hours in the cold
incubator.
9) Next lab, observe the plate agar to interpret the result.

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7) Incubate the nutrient agar plate in an inverted position
at 35°C to 37°C for at least 24 hours in the cold
incubator.
8) Next lab, observe the plate agar to interpret the result.

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 RESULTS & DISCUSSIONS

Escherichia coli Staphylococcus aureus

The results show isolated colonies of Escherichia coli and Staphylococcus aureus. Individual
or small groups of cells will divide producing a visible colony-forming unit, or colony. Observe
areas of well-isolated colonies. Different species will produce colonies that appear differently,
and these plates have the pure culture.

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 CONCLUSION

As conclusion, it was mandatory that aseptic techniques be followed when experimenting with
microorganisms. Compared to spread plate and pour plate techniques, streak plate was found
to be the most efficient and also the easiest way to isolate bacterial colonies. Pure cultures of
two types of bacteria that were obtained included Staphylococcus aureus (rod-shaped and
Gram-positive) and Escherichia coli (rod-shaped and Gram-negative). Other than that, the
importance of heat fixing and Gram staining was understood. Gram staining proved to be a
very good method for observing the bacterial cellular features and hence, differences between
Gram-positive and Gram-negative bacteria were clarified. It was also understood that reaction
of a bacterial cell to the Gram stain is determined by the decolourization step.

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 REFERENCES

Acharya Tankeshwar (2021). Streak Plate Method: Principle, Procedure, Uses. Retrieved from
https://microbeonline.com/streak-plate-method-principle-purpose-procedure-results/

Cynthia Ruscitto (2018). Isolation Techniques for a Streak Plate. Retrieved from
https://sciencing.com/isolation-techniques-streak-plate-8539650.html

Editor (2018). To study the isolation of pure cultures by using streak plate method. Labmonk.
Retrieved from https://labmonk.com/to-study-the-isolation-of-pure-cultures-by-
usingstreak-plate-method

Microbial Zoo (2020). Lab technique microbiology: Streak plate method. Retrieved from
https://www.youtube.com/watch?v=fND5I_A7wNM&t=66s

Sagar Aryal (2021). Streak Plate Method- Principle, Methods, Significance, Limitations.
Retrieved from https://microbenotes.com/streak-plate-method-principle-methods-
significance-limitations/

SJU GEP Science (2016). Streaking microorganisms on an agar plate. Retrieved from
https://www.youtube.com/watch?v=ku22XJc-c6c

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