Exercise 3

Preparation and Sterilization of Culture Media and Glasswares

Villarba, J., Virtucio, L., Wenceslao, B., Wepee, R., Yana, O.
2nd year 2Bio6

Introduction
Microorganisms need nutrients in
order to grow. Culture media, also called as
growth media, are nutrient solutions used in
laboratories to grow microorganisms. For
successful preparation of media of certain
microbes, understanding its nutritional
requirements and imparting the essential
nutrients in the proper amount and quantity
in a culture medium are necessary. They
can be in either liquid or solid form, the
former called Broth and the latter named
Agar. Liquid media are often mixed with
agar and poured into Petri dishes to solidify.
The process of killing or removal of all
microorganisms including bacterial spores
which are highly resistant is called
sterilization. Sterilization of culture media is
done by autoclaving, heating or membrane
filtration. If not done, contaminants may
grow and reproduce rapidly.
There are many ways to sterilize
culture media. One is through moist heating
and the other is through dry heating. Moist
heating includes boiling in a water bath and
steam under pressure. On the other hand,
dry heating utilizes hot air that is free from
water vapor but takes the process longer
since air is not an effective conductor of
heat.
In this experiment, we will learn that
there are different ways to prepare culture
media as well as sterilize it in different
manners. We will know what kind of

sterilization is more effective in terms of

accuracy and duration of time.

Objectives of the Experiment

This experiment aims to acquire and
develop the skills in the preparation of
media for the cultivation of microorganisms.
Specifically:
1. To learn how autoclaving and dryair oven works
2. To determine what type of
sterilization is more effective and accurate
3. To know the role of agar in media
preparation

Methodology
In this experiment, the following
equipments and materials were gathered:
15 grams Nutrient Agar (NA), 15 grams
Nutrient Broth (NB), 15g Potato Dextrose
Agar (PDA), 15g Potato Dextrose Agar
(PDB), 10 Petri dishes, 15 test tubes, two
Erlenmeyer flasks, calculator, triple beam
balance, microwave oven, aluminum foil
and an alcohol lamp. In order to prepare
the nutrient agar (NA) media, A triple beam
balance was used to weigh the nutrient
agar. A piece of aluminum foil shaped into a
boat was used to hold the powder while
weighing atop the pan of the triple beam

balance.
Then,
nutrient
agar
was
transferred to an empty Erlenmeyer flask
and 100mL of distilled water was added.
After suspending 15g nutrient agar in
distilled water, a microwave oven was
prepared and used until the agar was
dissolved in the flask. Meanwhile for nutrient
broth (NB) media, 5mL nutrient broth was
prepared and transferred into test tubes
using a serological pipette. The test tubes
and Erlenmeyer flasks were labeled
appropriately. Then, cotton plugs are then to
be placed on the test tubes and Erlenmeyer
flasks containing media.
Glasswares
and
media
were
sterilized separately at 121 degrees Celsius
for 15 to 20 minutes in the autoclave and
were dispensed. After sterilization, each
three NA and PDA slant tubes were cooled
in an inclined position until the medium
solidified while the three NA deep tubes are
cooled in an upright position. For the plated
media, flasks were cooled to around 45 to
50 degrees Celsius. 15 to 20 mm of medium
were aseptically poured into each sterile
plate and solidified. The lid of the Petri dish
was sterilized first by an alcohol lamp and
was lifted while simultaneously pouring the
medium. The media was used after the
media have already solidified.

Results and Discussion

This figure shows how the autoclave

in the microbiology laboratory works. An
autoclave needs water to make the steam
used for sterilization. Smaller models will
have a reservoir that requires you to refill it
before use, as with a coffeemaker. Large
standing models, such as those used in
laboratories, will have a water intake
hookup or hose, allowing the user to pump
water directly into the machine. The
chamber is the space where the user places
items to sterilize. In the chamber are wire
racks, like those in a dishwasher, which will
hold various items upright or lying down and
allow for steam penetration from all angles.
The control panel allows the user to
customize the autoclaving process. Some
materials
can
withstand
higher
temperatures, while some must be
autoclaved at lower temperatures for longer
time. The heat, pressure and time will vary
with each type of item autoclaved, creating
the need for a control panel. Some
autoclaves will include automated settings
for specific autoclaving functions; these
models will have "fast buttons" for those
settings. Autoclaves must have an air pump
system to remove the oxygen in the
chamber and create a vacuum which then
fills with pressurized steam created from the
water in the reservoir. The water becomes
heated either via a heating element inside
the water reservoir or a heat-generating
mechanism that completely and accurately
surrounds
the
reservoir.
[1]

1. How does autoclaving and dry-air

oven works?
2. What type of sterilization is more
effective and accurate?
Moist sterilization which includes steaming
acts by denaturation and coagulation of
protein, breakage of DNA strands, and loss
of functional integrity of cell membrane. is

more effective than dry heat by these

reasons:
a. Bacteria are more susceptible to moist
heat

concluded that moist heating is more

effective than dry heat. Lastly, the usage of
autoclave and dry-air oven is important to
learn when culturing media.

b. Steam has more penetrating power

c. Steam has more sterilizing power as
more heat is given up during condensation
[2]

References
All About Agar. (2002). Retrieved from
http://www.sciencebuddies.org/scien

3. What is the role of agar in media

preparation?
Agar is a gelatinous substance
obtained from various kinds of red seaweed
and used in biological culture media and as
a thickener in foods such as ice cream and
other desserts. It is a phycocolloid extracted
from a group of red-purple marine algae
(Class
Rhodophyceae)
including Gelidium, Pterocladia and Gracilar
ia.Gelidium is the preferred source for
agars. During media preparation, agar is a
solidifying agent and without it, the culture
remains liquid. It remains firm at
temperature as high as 65 degrees Celsius
and used in a final concentration of 1-2% for
solidifying culture media.

ce-fairprojects/project_ideas/MicroBio_Ag
ar.shtml
Martinko, M. (2005). Brock Biology of
Microorganisms (11th ed.). Prentice Hall
Inc: USA
Mitchell, K. (2009). Parts and Functions Of
An Autoclave. Retrieved from
http://www.ehow.com/info_8611075
_parts-functions-autoclave.html

Conclusion
Based on the data and results, the
researchers therefore concluded that
sterilization and autoclaving are necessary
when preparing culture media because
failure performing it will have a 100% of
contamination. The researchers also

Sterilization And Disinfection. (2010).

Retrieved from
http://generalbacteriology.weebly.co
m/sterilization-and-disinfection.html