1.

0 Introduction

During doing an experiment, cleanliness is the most important thing to take care off
because it is to ensure that we can get the exact result that we want from the
experiment. Thus, this technique is called aseptic techniques. Aseptic technique,
designed to provide a barrier between the microorganisms in the environment and the
sterile cell culture, depends upon a set of procedures to reduce the probability of
contamination from these sources. 1 The researchers also said the elements of aseptic
technique are a sterile work area, good personal hygiene, sterile reagents and media,
and sterile handling.

Failure on practicing aseptic technique, does not sterile the equipment and the
work surface will lead to many undesired result especially in biological
contamination. This is because microorganism only can be spread and live on the
normal temperature of that place. That is why we need to culture new environment
that will prevent the microorganism to affect our experiment.

Throughout the experiment, every activity like transferring the sample need to
be done using the aseptic technique which is these experiment all the activity was
done near to the flame. From this action we can make sure that there will be no effect
from the environment to the sample.

1.1 Objective
a) To teach on how to maintain the sterility during microbiological work.
b) To teach on methods and techniques for aseptic techniques.

1
http://www.lifetechnologies.com/my/en/home/references/gibco-cell-culture-basics/aseptic-technique.html

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2.0 Experimental Methodology
2.1 Apparatus
1. A bottle of ethanol at 75% concentration
2. 2 bottles of sterile molten agar
3. 2 flasks containing nutrient broth
4. 2 sterile Petri dishes and 2 non-sterile Petri dishes
5. Bunsen burner (to be placed in laminar flow cabinet)
6. 2 inoculating loops
7. Sterile pipettes and tips
8. A bottle of sterile distilled water
9. Laminar flow cabinet
10. Incubator shaker

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 2.2 Procedure

Procedures with
Aseptic Techniques

Table is wiped with alcohol

The Bunsen burner is lit on

Apparatus is arranged close to the Bunsen burner and allowed for 10 minutes before start to
work.

Inoculating loop is placed in the alcohol solution

Molten agar is poured aseptically into an empty Petri dish and lid is closed quickly and the
agar is allowed to harden

0.1ml of distilled water is aseptically dispensed and transferred into the nutrient broth

Wire loop is flamed until red hot and cooled down in air

The loop is dipped into the distilled water and an S-shape is streaked aseptically on the
surface of the agar

Lid of the Petri dish is closed quickly

Agar plates and nutrient broths kept in incubator for 24h at 37◦C

Table top is wiped after completing the work and all the apparatus is removed

Growth of microorganisms in the agar and broth is observed

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 Procedures without Aseptic
Techniques

Work is performed on the bench in the laboratory

Required apparatus is arranged on the table

Molten agar is poured into an empty Petri dish by allowing the agar to flow across the dish

Lid of Petri dish is closed and agar is allowed to harden

0.1ml of distilled water is dispensed and transferred into the nutrient broth

Loop is dipped into the distilled water and an S-shape is streaked on the surface of the agar

The lid of the Petri dish is closed quickly

Agar plates and nutrient broths kept in incubator for 24h at 37◦C

Growth of microorganisms in the agar and broth is observed.

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 3.0 Results and Discussion
3.1 Results

Aseptic techniques Without aseptic techniques

Nutrient broth Nutrient agar Nutrient broth Nutrient agar
Growth (+) (-) (-) (-) (+)
or (-)

Draw the
distribution
of growth

Figure 3.1.1: Figure 3.1.2: Figure 3.1.3: Figure 3.1.4:

Aseptic Aseptic Non-aseptic Non-aseptic

Technique Technique Technique Technique
Universal Petri Dish 1 Universal Petri Dish 3
Bottles bottles

Observation Clear Clear Clear White patches

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3.2 Discussion

Hypothesis of this experiment should clarify that by using aseptic technique,

there will be no growth of any microorganism. Observation at the end of the
experiment showed that the hypothesis is true where there were no growth of any
microorganism in both nutrient agar and nutrient broth through aseptic technique.
The clear solution in Figure 3.1.1 for nutrient broth and also clear surface for nutrient
agar can be seen in the Figure 3.1.2 explains theory. This is because, through aseptic
technique, the dipping of the distilled water into the nutrient broth and nutrient agar
were done near the flame which destroys microorganisms as it cannot withstand high
temperature. Moreover the sterile of petri dish and tips that were used also helps to
render sterility of the technique.

According to the hypothesis, there are chances for contamination in the

nutrient samples while practicing lab without aseptic technique. Nutrient broth in
Figure 3.1.3 showed clear solution where cloudy form signifies contaminated sample.
While petri dish No.3 of nutrient agar in Figure 3.1.4 resulted with small white
patches on the agar. There are some reasons on why there was no growth of any
microorganism in the nutrient broth. Firstly the small surface contact of the universal
bottles compared to the petri dish shows higher risk in exposed to the surrounding
condition. Moreover, the tip that has been used during the practice is already sterile
which prevents from getting contaminated. Petri dish has growth because it has higher
surface contact to the surrounding which resulted in higher possibility to expose to
other microorganism. Inoculating loop was bent using hand before dipped into the
distilled water which might cause contamination. Even, while dipping the loop into
distilled water also exposed to surrounding condition. So, there was growth of
microorganism when practicing experiments without aseptic technique.

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4.0 Conclusion

Based on the above reasons, it can be concluded that the objective is

Successfully achieved. There is no growth of any microorganism in the nutrient agar
or broth when using the aseptic technique. Without aseptic technique growth of
microorganisms on the nutrient agar is observed but not in nutrient broth which is
clear as initial condition. By the end of this experiment, students learnt on how to
perform microbiological work by maintaining the laboratory condition, methods and
techniques which will render sterility called aseptic technique. This technique
improves microbiological work which is to identify growth of only desired pure
culture and not affected with other microorganisms. Observation supports the
objective of this experiment. It is highly recommended to use aseptic technique to
perform such microbiological work in order to enhance accuracy in findings.

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5.0 References

[1] Bauman, Robert W. Microbiology San Francisco: Pearson Education Inc., 2004.

[2] Betty, S., Boone, D., Fletcher, D. The effects of aseptic and sterile technique on
contamination in microbe cultures. Final Research., 2009.

[3] Retrieved from http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm