The added to promote plant’s growth whereas the carbon

            The culture media used for in vitro cultivation of
explants are composed of complex mixture of salts, vitamins, carbon source,
solidifying agents, plant growth regulators and antibiotics. The complex
mixture of salts is divided into three groups which are macroelements, microelements
and iron source. The examples of macroelements are N, K, P, Ca, S and Mg and
they are building blocks of amino acid, proteins, nucleic acid, cell wall and enzyme
cofactors. However, the micronutrients like zinc, copper and cobalt are usually
part of certain enzymes. The vitamins usually added to promote plant’s growth
whereas the carbon source is needed as the explants have insufficient chlorophyll
or maturity to produce food by itself. The carbon source that commonly used is
sucrose which is readily assimilated and relatively stable. The
solidifying agent in this experiment is agar. It plays supporter role
for cultures and it has many advantages such as it does not react with media
constituents, it does not digested by plant enzymes and it can remain stable at
all incubation feasible temperature. The antibiotics that prevent growth of
microbes are not used because it may inhibit cell growth that is why in the
first week of observation there is already contaminated by fungus.

            There is several precaution steps needed to be carried
out during experiment. For examples, the working areas and equipment that should be
sterilized with alcohol such as 70% ethanol solution before performing the
tissue culture. These protocols describe basic procedures for aseptic technique
in cell culture technology. Firstly, personal hygiene is the one basic concern
for a successful aseptic technique. This is because human skin harbors a
naturally occurring and vigorous population of bacterial and fungal inhabitants
which is shed microscopically and ubiquitously on ours skins which may
contaminated ours experiments. Most unfortunately for cell culture work, cell
culture media and incubation conditions provide ideal growth environments for
these potential microbial contaminants. This procedure outlines steps to
prevent introduction of human skin flora during aseptic culture manipulations. Every
item that comes into contact with a culture must be sterile in order to avoid
any contamination on ours experiments. This includes direct contact as well as
indirect contact too. Ideally, all aseptic work should be conducted in a
laminar cabinet .However; work space preparation is essentially the same for working
at the bench. Flame sterilization is used as a direct, localized means of
decontamination in aseptic work at the open bench. It is most often used to
eliminate potential contaminants from the exposed openings of media bottles,
culture flasks, or test tubes during transfers. Besides, to sterilize small instruments
such as forceps or to sterilize wire inoculating loops and needles before and
after transfers. Where possible, flame sterilization should be minimized in
laminar-flow environments as the turbulence generated by the flame can
significantly disturb the sterile air stream.

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            In this experiment, cutting methods also the things that
we need to concern about in order to avoid contamination and to make us easy to
handle the garlic embryos. This is because in laminar flowhood there is not
enough space for us to cut the embryo like we cut the garlic in the kitchen. So,
we must cut the garlic in order to get the embryo by using forceps and scalpel.
The positions of the garlic must be horizontal because it is easier to dissect
the garlic in horizontal positions. When the garlic is cut in horizontal
positions, it is easy and more comfortable for us to cut and avoid from the
garlic to jump out from the paper that we use to cover up the laminar flowhood
floor. On the other hand, our lab coat cannot touch the laminar flowhood when
cutting the garlic embryo because it may conduct a contamination if our lab
coat touches the floor.

            From the results above, the culturing explants in petri
dishes are contaminated with fungus from the first week but the explants in
culture vials (callus induction) are not contaminated with any fungus or
bacteria. The reason is due to the different source of explants. The source of
former is from outside plantlet while the latter is from the in vitro plantlet.
Hence, although the outside plantlet’s was washed and sterilized with Clorox and
Tween 20, there are may be some dusts or spores hidden inside the cracks of
plant leaf. In contrast, the in vitro was already in aseptic condition,
therefore there will be no contaminants inside the vial.