3.

0 RESULTS
Aseptic technique

Figure 3.1 Aseptic Agar

Figure 3.2 Aseptic broth

Non aseptic technique

Figure 3.3 Non Aseptic Agar

Figure 3.4 Non Aseptic Broth

Figure 3.5 Streaking plate
DISCUSSIONS

The objectives of this experiment is after completing this laboratory, students are able to prepare
microbiological media by own and also students will be exposed to media preparation and
aseptic technique involve during media preparation.

There are many types of culture media and in this experiment we are using two types of culture
media which are nutrient agar and nutrient broth. “The only difference between broth and agar
media is that broths do not contain an agar component. We use broth tubes primarily for specific
assays, or (rarely) for bacteria that will not form colonies on a solid surface.” (Howler, 2012).

The methods used in this experiment are aseptic and non-aseptic media. “Aseptic techniques
must be used to reduce the likelihood of bacterial contamination. This usually involves
disinfection of working areas, minimizing possible access by bacteria from the air to exposed
media, and use of flames to kill bacteria which might enter vessels (petri dish or bottle) as they
are opened” (David R. Caprette, 2011). On the other hand, non-aseptic technique is an open
bench procedure without any contamination prevention method.

The result must be observed 48hours after the experiment. Based on the result that we observed
for aseptic method, the nutrients agar in the petri dish and nutrient broths, there are no formation
of bacterial growth or they are not contaminated at all. As an example of a typical transfer, these
are the steps required for good sterile technique in transferring bacteria from one capped test tube
to another. Note that once the transfer is started, nothing is layed on the bench top and everything
is flamed before and after. First, flame the inoculating loop and then flame the mouth of the tube
while holding the cap. Next, remove some bacteria with the loop, and flame the mouth of the
tube and replace the cap. After that, flame the mouth of the tube to which the bacteria are being
transferred. Inoculate the tube with bacteria from the loop then flame the mouth of the tube and
replace the cap. Lastly, flame the inoculating loop.

For non-aseptic method, the result that we observed nutrient agar in the petri dish and nutrients
broths, the nutrient agar have contain bacterial contamination. For nutrients broths, the sample
also contain bacterial contaminations. It was said bacterial contamination due to the formation of
residue inside the bottles and a visible patch form on the agar inside petri dish.
For the aseptic method, theoretically there shouldn’t be any bacterial contamination inside the
media because this method is used to reduce any bacterial contamination inside the culture
media. These methods are involving disinfection of working areas and use flame as a prevention
of bacteria from the surrounding are being exposed to the culture media because these bacteria
will contaminate the media. Moreover, before we started this aseptic method, we need to sterile
these two culture media by using autoclave. “An autoclave is designed to deliver steam into a
pressure chamber, generating high heat and pressure at the same time. Heating media to above
121 degrees C for 4 to 20 min destroys nearly all living cells and spores” (David R. Caprette,
2011).

Based on result that we obtained from the aseptic method, there’s no contamination that
observed inside the media. So, we are successfully achieved the objectives of preparing media by
aseptic technique.

Based on result for non-aseptic method, theoretically there’re should be bacterial contamination
inside the nutrient agar and nutrient broth. This is because non-aseptic method will exposed
bacteria in the air directly to media as it hasn’t any prevention bacterial contamination procedure,
like aseptic method inside this method.

A common microbiological technique for the isolation of pure bacterial cultures is the streak-
plate technique. This technique allows one to easily separate individual bacterial cells on a solid
medium. As we will see, this technique is essential for isolating clonal populations of bacteria
and, through the observation of the resulting bacterial colonies, is an important component of
bacterial identification. Basically, this technique involves streaking a loopful of bacteria across a
solid medium, such that individual bacterial cells fall off the loop and are deposited on the
medium. Each individual cell, then, will replicate in that location, giving rise to a colony of
genetically identical cells - a clonal population. As you probably can imagine, this technique can
be used for many purposes, from separating different species growing in a mixture to
determining how many bacterial cells are present in a solution.

For streaking plate, generally, bacteria are obtained by sticking a sterile loop into the bacterial
culture (either a liquid culture, a culture growing on a solid medium, or an 'environmental'
culture) . This loop then is streaked repeatedly across about 1/3rd of the plate. The plate then is
rotated and a new, sterile loop or needle is used to spread some bacteria from the primary region
across another 1/3rd of the plate. Finally, this step is repeated for the final 1/3rd of the plate. The
result obtained was that many bacteria are deposited in the primary region, fewer bacteria are
deposited in the secondary region, and even fewer bacteria are deposited in the final region.
These plates then are incubated, allowing the individual cells to replicate and give rise to
colonies. Ideally, the plate will contain many colonies in the primary region (perhaps even a
confluent lawn of bacteria) and well-isolated, individual colonies in the final region.

In this experiment, after we had prepared both culture media for microbial growth and before we
incubated these media, we need to invert the Petri dishes as to prevent condensation droplets
from falling onto the surface of the agar and get a better result for bacterial contamination
observation.
4.0 CONCLUSIONS

From the result and the discussion above, theoretically in aseptic method there shouldn’t be any
microbial growth activities inside the petri dish because technically the experiment was
conducted in a controlling workplace rather than in non-aseptic method. On the other hand,
conducting the experiment in a non-controlling workplace and encouraged the microbial growth
activities inside the petri dish is what being called as non-aseptic method. Based on result for
non-aseptic method, theoretically there are bacterial contamination inside the nutrient agar and
nutrient broth. This is because non-aseptic method will exposed bacteria in the air directly to
media as it hasn’t any prevention bacterial contamination procedure, like aseptic method inside
this method. For streaking plate, the result obtained was that many bacteria are deposited in the
primary region, fewer bacteria are deposited in the secondary region, and even fewer bacteria are
deposited in the final region. In nutshell, the objectives are achieved.
5.0 REFERENCES

1) Martin, S. (n.d.). Sterile Technique. Retrieved October 27, 2014.

2) Anonymous. BIO 302: Lab 1. (2012). Retrieved October 27, 2014.