LAB 3 :
PREPARATION AND STERILIZATION OF CULTURE MEDIA
Name : Kong
Siao Thung
Matrice No.
: 111372
Introduction
Culture media are available
commercially as powders ; they require only the addition of water. Nutrient
medium is a general purpose preparation for culturing microorganisms which are
not nutritionally fastidious. The broth contains:
3.0 g/L “Lab-lemco”
powder ( a beef extract)
2.0 g/L yeast extract
5.0 g/L peptone (a
nitrogen source)
5.0 g/L sodium
chloride
15.0 g/L agar powder
The agar has the same
composition, except that it contains 15 g/L agar. The final pH of both media is
7.4.
Autoclaving is a process that
use moist heat and pressure so that all parts of the material to be sterilized
reach 121oC for 15 minutes. An autoclave is, in essence, a large
pressure cooker; a chamber which may be sealed off against surrounding air.
Materials for sterilization are placed in the chamber, the door is sealed, and
pressurized steam is forced into the chamber. The incoming steam displaces
cooler air through an exhaust valve; this valve closes when the cell cooler air
has been vented.
Steam is continually forced into
the chamber until the pressure reaches
103 kPa above atmospheric pressure; at sea level, this pushes the temperature
in the chamber to 121oC throughout. After sterilization, the steam
pressure is slowly decreased to atmospheric pressure. The sterilized objects
can then be removed.
Objective
To prepare sterile
nutrient agar for culturing microorganisms
Discussions
a) We
have weighed appropriate amount of broth(with agar) powder into Scott bottle
and dissolved it with distilled water. Then, we have mixed it well using a glass
rod and boiled it.
b)
After
the solution is boiled, we loosely recap the bottle and set aside for
sterilization. The media needed to be sterilized at 121oC for 15
minutes.
c)
We
used autoclave method to sterilize the media.
d)
An autoclave is an instrument used to sterilize equipment and supplies by
subjecting them to high pressure saturated steam at 121 °C for around
15–20 minutes depending on the size of the load and the contents.
e)
Autoclaving is the process of sterilization by
pressure, heat, and chemical agents to disinfect materials.
f))
After
autoclaving, we removed the media and allowed the broth preparation to cool
then tighten the cap of each bottle.
g)
Laminar flow
provides clean air to the working area and provides the constant flow of air
out of the working area to prevent room air from entering.
h)
The air
flowing out from the hood suspends and removes contaminants introduced into the
work area by personnel.
i)) Laminar flow
has a high efficiency bacteria-retentive filter. Room air is taken into the
unit and passed through a pre-filter to remove gross contaminants. The air is
then compressed and channeled up behind and through the HEPA filter in a
laminar flow fashion. The HEPA filter removes nearly all the bacteria from the
air.
Conclusions
To prepare a sterilized culture media, the
quantity of broth need to be précised and the aceptic technique which is the
autoclaving is the most effective way of
sterilization.
References
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