DIPLOMA IN PHARMACY COHORT 35

YEAR 3 SEMESTER 1

PAPM 321.2 PHARMACEUTICAL MICROBIOLOGY (P)

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LAB REPORT 1 : ASEPTIC TECHNIQUE (TRANSFER OF BACTERIA FROM

ONE MEDIA TO ANOTHER MEDIA)
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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

In the field of microbiology, aseptic technique is a fundamental and important laboratory skill.
It is also very compulsory laboratory skill to conduct research related in the field of
microbiology. Aseptic technique can define as a proper method that involves target-specific
practices and procedures under suitably controlled conditions to prevent the contamination of
cultures from microbes inherent in the environment. For example, airborne microorganisms
(including fungi), microbes picked up from the researcher’s body, the lab bench-top or other
surfaces, microbes found in dust, as well as microbes found on unsterilized glassware and
equipment may potentially contaminate cultures, thus interfering with the lab results.
Transferring cultures, inoculating media, isolating pure cultures, and performing
microbiological tests are all procedures that microbiologists use aseptic technique for.
Therefore, for in this Lab Report 1, we will learn how to subculture bacteria by using a variety
of culture media as our inoculate sources and as our new culture media. Species of bacteria
such as Escherichia coli and Staphylococcus aureus will be used, so that we can become
familiar with different growth patterns.

Escherichia coli Staphylococcus aureus

OBJECTIVES

1) To transfer cultures from one medium by inoculating another medium (subculturing).

2) To eliminate potential contamination of bacterial cultures and media from microbes in

the environment by using aseptic technique.

3) To identify different ways by which bacteria grow in culture – on agar slants, on agar
plates, and in broths.

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 MATERIALS

Inoculating Loop

Bunsen Burner

Test Tube Rack

Nutrient Agar Plate

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 Nutrient Broth

Nutrient Agar Slant

Sample of Escherichia coli

(Gram-negative)

Sample of Staphylococcus aureus

(Gram-positive)

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 METHODS / PROCEDURE

PROCEDURE FOR INOCULATING A NUTRIENT BROTH TO NUTRIENT AGAR PLATE

1) Sterile the inoculating loop by flaming it by using bunsen burner until red-hot, and allow it to cool.

2) Remove the lid of the tube (nutrient broth sample) using the little finger. Avoid using thumb and never place
the lid onto the table.

3) Sterilize the tops of the tube by passing the tube’s mouth through the flame. This is to kill any airbone microbes
that may contaminate the tube entrance during transfer.

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4) Dip the inoculating loop into the liquid culture (nutrient broth sample). Then, carefully remove the loopful of
inoculum without touching the rim of the tube.

5) Sterilize the tops of the tube by passing the mouth through the flame again before replacing the lid.

6) Lift the lid of the plate and hold it over the base as a shield to prevent contamination from falling onto the
plate. Then, transfer the bacteria to petri plate (nutrient agar plate) by spreading it over the entire surface of
the plate in all directions. Rotating the plate a quarter turn between spreads to ensure that the bacteria is spread
evenly across the plate.

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7) Sterile again the inoculating loop by flaming it by using bunsen burner until red-hot.

8) Lastly, incubate the nutrient agar plate that have been inoculated at 37℃ - 38℃ for 24 hours.

9) Next lab, observe the nutrient agar plate to interpret the result.

PROCEDURE FOR INOCULATING A NUTRIENT BROTH TO ANOTHER NUTRIENT BROTH

1) Sterile the inoculating loop by flaming it by using bunsen burner until red-hot, and allow it to cool.

2) Remove the lid of the tube (nutrient broth sample) using the little finger. Avoid using thumb and never place
the lid onto the table.

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3) Sterilize the tops of the tube by passing the tube’s mouth through the flame. This is to kill any airbone microbes
that may contaminate the tube entrance during transfer.

4) Dip the inoculating loop into the liquid culture (nutrient broth sample). Then, carefully remove the loopful of
inoculum without touching the rim of the tube.

5) Sterilize the tops of the tube by passing the mouth through the flame again before replacing the lid.

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6) Then, take the another tube of liquid media (new nutrient broth). Open the lid of the tube and pass the mouth
through the flame.

7) Dip the inoculating loop with bacteria into the tube, and carefully remove it. Flame again the mouth of the
tube before replacing the lid.

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8) Sterile again the inoculating loop by flaming it by using bunsen burner until red-hot. Then, place the tube
(contain nutrient broth) into the tube rack.

9) Lastly, incubate the tube (nutrient broth) that have been inoculated at 37℃ - 38℃ for 24 hours.

10) Next lab, observe the nutrient broth to interpret the result.

PROCEDURE FOR INOCULATING A NUTRIENT AGAR SLANT TO ANOTHER NUTRIENT

AGAR SLANT

1) Firstly, labelling the tube of nutrient agar slant (sample) and another nutrient agar slant (new media). Label it
with initials of the bacteria and the date.

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2) Hold the inoculating loop like holding a pencil in dominant hand, and then sterile the inoculating loop by
flaming it using bunsen burner until red-hot. Next, allow it to cool.

3) Pick up the bacteria sample, open the cap, and then flame the mouth tube area to sterilize.

4) Tilt the tube, put it an angle and take the sample. After that, flame the mouth tube area and put the cap back
on.

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5) Now, to transfer the bacteria, take the another agar slant tube, open the cap, and flame the mouth area tube.

6) Insert the inoculating loop that already has bacteria towards the base of the slant. Make sure the inoculating
loop not touch the mouth tube.

7) Pull out the inoculating loop in zig zag way up, and then flame the mouth tube to sterilize, and put the cap
back on. Next, flame the inoculating loop to sterilize.

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8) Close the tube (that have been inoculated) properly.

9) Lastly, incubate the tube (nutrient agar slant) that have been inoculated at 37℃ - 38℃ for 24-48 hours.

10) Next lab, observe the nutrient agar slant to interpret the result.

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 RESULTS & DISCUSSIONS

RESULTS

TYPE OF
Nutrient Broth Nutrient Agar Slant Nutrient Agar Plate
NUTRIENT
TYPE OF Staphylococcus Staphylococcus
Escherichia coli
MICROORGANISM aureus aureus

Figure 5.2
Figure 5.1

Figure 5.3

FIGURE OF
RESULT

OBSERVATION Sterile Sterile Sterile

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 DISCUSSIONS

TYPE OF
Nutrient Broth
NUTRIENT
TYPE OF
Staphylococcus aureus
MICROORGANISM

Figure 5.1

FIGURE OF
RESULT

Observation of Staphylococcus aureus turbidity flocculent. The presence of Staphylococcus

INTERPRETATION aureus makes the liquid medium (nutrient broth) cloudy. This may be the result of only one
bacterial cell first entering the medium and then repeatedly dividing to produce millions.

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 TYPE OF
Nutrient Agar Slant
NUTRIENT
TYPE OF
Staphylococcus aureus
MICROORGANISM

Figure 5.2

FIGURE OF
RESULT

Staphylococcus aureus showed luxuriant growth on nutrient agar slant. Growth on agar can
INTERPRETATION
be seen by noting the appearance of a thick.

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 TYPE OF
Nutrient Agar Plate
NUTRIENT
TYPE OF
Escherichia coli
MICROORGANISM

Figure 5.3

FIGURE OF
RESULT

E. coli colony appears in round/circular shape with off-white or beige in colour with a shiny
texture. It often looks like mucus or a cloudy film over the whole surface of the plate. E.
INTERPRETATION coli colony is slightly raised and has an entire, fixed margin and a steady growth pattern,
creating concentric growth rings in the colony. These rings can be detected under a
microscope. Older colonies often have a darker center.

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 CONCLUSION

In conclusion, aseptic technique is of utmost importance to maintain pure stock cultures while
transferring cultures to new media. It is because using proper aseptic technique can greatly
minimize or even eliminate the risk of contamination. Aseptic technique is also essential for
isolation of a single species of microorganism from a mixed culture to obtain a pure culture.
Thus, proper aseptic technique prevents microbes used in the laboratory from accidentally
being released into the environment and infecting people working in the laboratory. This is
especially relevant when pathogens are being handled. Therefore, aseptic techniques usually
involve disinfection of working areas, minimizing possible access by bacteria from the air to
exposed media and use of flames to kill bacteria that might enter vessels as they are opened.

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 REFERENCES

Bio-Rad Laboratories (2012). Aseptic Technique. Retrieved from

https://www.youtube.com/watch?v=bRadiLXkqoU

Buddies, S. (2020). Interpreting Plates. Science Buddies. Retrieved from

https://www.sciencebuddies.org/science-fair-projects/references/interpreting-agarplates

Dr. Julie Wells (2020). Lab 1-3: Aseptic technique. Retrieved from
https://www.youtube.com/watch?v=v0G8Hd6R-14

Fujikawa, H., & Morozumi, S. (2005). Modeling Surface Growth of Escherichia coli on Agar
Plates. Applied and Environmental Microbiology, 71(12), 7920–7926. Retrieved from
https://doi.org/10.1128/aem.71.12.7920-7926.2005

Nhut Tran (2016). Aseptic Technique. Microbiology Lab Manuals. Retrieved from
https://www.slideshare.net/NhutTran8/2015-mibi-labmanual

Richard Steane (2016). Experiment to show the growth of the bacteria-basic technique.
BioTopics. Retrieved from https://www.biotopics.co.uk/microbes/tech.html

Sangwan, A. (2016). Escherichia Coli (E. Coli): Meaning, Morphology and Characteristics.
Biology Discussion. Retrieved from
https://www.biologydiscussion.com/bacteriology/systematic-
bacteriology/escherichiacoli-e-coli-meaning-morphology-and-characteristics/30821

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