Basic techniques in microbiology: Aseptic technique and inoculation.

Objectives
The objectives of this laboratory experiment are:
1. To acquire basic skill of aseptic technique in the field of Microbiology.
2. To prevent contamination of cultures and media from microbes in the environment.
3. To transfer cultures from one medium by inoculating another medium (subculturing).
4. To acquire proper aseptic technique in the streak plate technique.
Results

Medium Result
Solid Quadrant streaking
cultured
medium
(NA)

Simplified streaking (zig-zag

pattern)

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 Liquid Streak-slant agar
cultured
medium
(NB)

Stab-flat agar

Table 1 : Data of cultures

Observation
After an hour incubate the nutrient broth at 370C, the broth appeared to be turbid. This
indicates that there are bacterial growth.
After 24 hours of incubation at 370C, whitish spots are observed in all agars based on
Table 1. In quadrant streaking agar, distinct translucent circular single colonies is observed in
the last streaking quadrant. While in the simplified streaking, translucent circular colonies also
are observed but lesser than the streaking agar. For the liquid cultured medium, streak-slant
agar has translucent circular colonies on its surface and has more colonies compared to stab-
flat agar. While the stab-flat agar, very few colonies are only observed in the stab area.
Question
1. Did something grow in all of your transfers? If not, what could have been the problem?
Yes, there are growth in all of the transfers.

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 2. Did the quadrant streak method produce single colony?
The quadrant streak method give better distinct single colony compared to simplified
streak method. Single colonies can be observed in the last streaking quadrant.

3. Do any of your agar plates appear to be contaminated?

No, all of the agar plates are aseptically preserved. This is because 70% of ethanol is
used to disinfect the working area to kill unwanted microbes. Besides, the inoculation
is conducted properly and good precaution is practiced such as no talking during the
inoculation to prevent unwanted microbes from our saliva to contaminate the agar. The
inoculating loop also is well heated and the mouth of bottle is well pass through the
flame.

4. How would you know if your broth cultures were contaminated with a microbe other
than the one you wanted to grow?
If the broth cultures were contaminated with unwanted microbes, there will be no
growth on the cultures such as the plate looks exactly the same as it did on the day on
inoculation, or there will be growth but various type of colonies grow in the cultures.
Possible contaminant during the inoculation is actinomycetes that can contaminate the
cultures and produces spores.

Discussion
Briefly discuss your findings and relate the importance of these techniques in pharmaceutical
industry settings.

The importance of streaking techniques is to grow bacteria on an agar plate and isolate
individual bacterial colonies. Isolated colonies indicate a clone of cells, being derived from a
single precursor (Centers, 2015). From the experiment, a pure culture is obtained as the growth
of microbe on the agar surface is similar to one another colony.
Stab-flat agar is used to inoculate semi-solid medium for the analysis of motility or
oxygen usage (Koneman, D., Janda, Schreckenberger, & Winn W. C., 2006). Through this type
of agar, it allows to culture a microorganism under limited oxygen usage. While streak-slant
agar provides large surface area, on which the microbes can grow and it give more oxygen
exposure compared to stab-flat agar.

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 Simplified streaking (zig-zag pattern) is usually performed when sample does not have
a high cell density. Based on the experiment, this type of agar did not give a better isolated
colonies as the bacteria used has high cell density. While quadrant streaking gives better
isolated colonies that can be seen in the last quadrant. This is because streaking in successive
areas of plate, the inoculum is being diluted for every few milimetres thus isolated colonies can
be obtained . An isolated colony is formed when these lone bacteria cells divide and give rise
to thousands of new cell (Acharya, 2016)
During the experiment, aseptic technique is highlighted, this is because to avoid
contamination of cultures and also to protect the students from getting infection from the
samples. Few precaution steps are practiced during experiment in order to get better isolated
colonies such as using a small amount of inoculum, the sample is lightly streaked so that it will
not gouge the agar and also the loop is flamed after streaking each quadrant. During incubation
of the cultures, the plate agar is placed side-up. This is because in order to prevent condensation
from dropping into the cultures as water can be the chemical contaminant

Most of the antibiotic that available today are from various microbes. Bacteria is
preferably chosen because they are easy to isolate, culture, maintain and to improve their strain.
Thus in order to produce a good antibiotic, mastering these techniques is crucial in
pharmaceutical industry.

Summary
From this experiment, aseptic technique and streaking are important techniques that
students should learnt and practiced. Successful cell culture solely depends on keeping the
microbes free from contamination by other unwanted microbes and also chemical contaminant.
Besides, good streaking can produce nicely isolated colonies of microbes on an agar plate as it
is very crucial to separate microbes in a mixed culture in order study the colony morphology
of an organism.

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References
Acharya, T. (2016). Streak plate method: Principle, Purpose, Procedure and results -
microbeonline, (April), 1–5. Retrieved from https://microbeonline.com/streak-plate-
method-principle-purpose-procedure-results/
Centers, N. (2015). Streak Plate Method Objective : Principle :, (2), 3–4.
Koneman, E. W., D., A. S., Janda, M. W., Schreckenberger, P. C., & Winn W. C. (2006).
Introduction to Microbiology. In: Color Atlas and Textbook of Diagnostic
Microbiology., 67–110. https://doi.org/doi:10.1201/9781420021622.ch1