Exercise 3Preparation and Sterilization of Culture Media and
GlasswaresVillarba, J., Virtucio, L., Wenceslao, B., Wepee, R.,
Yana, O.2nd year 2Bio6
IntroductionMicroorganisms 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 ExperimentThis 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
dry-air oven works2. To determine what type of sterilization is
more effective and accurate3. To know the role of agar in media
preparation
MethodologyIn 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 Discussion1. How does autoclaving and dry-air oven
works?
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]
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 heatb. Steam has more penetrating powerc.
Steam has more sterilizing power as more heat is given up during
condensation [2]
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)
includingGelidium,PterocladiaandGracilaria.Gelidiumis 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.
ConclusionBased 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
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.
ReferencesAll About Agar. (2002). Retrieved from
http://www.sciencebuddies.org/science-fair-
projects/project_ideas/MicroBio_Agar.shtmlMartinko, M. (2005).Brock
Biology of Microorganisms(11th ed.). Prentice Hall Inc:
USAMitchell, K. (2009). Parts and Functions Of An Autoclave.
Retrieved from
http://www.ehow.com/info_8611075_parts-functions-autoclave.htmlSterilization
And Disinfection. (2010). Retrieved from
http://generalbacteriology.weebly.com/sterilization-and-disinfection.html
