LAB
3: PREPARATION AND STERILIZATION OF CULTURE MEDIA
Introduction
Culture media provides vital
nutrients for microorganisms or cells to grow. In general, every culture media
provides specific nutrients which are suitable for different microorganisms or
cells. There are many types of culture media, for example commercial nutrient
agar, Brain Heart Infusion Agar (BHI) and Trypticase Soy Agar with 0.6% Yeast
Extract (TSAYE).
The self-made nutrient broth agar contains:
3.0 g/L “Lab-Lemco”
powder (beef extract)
2.0 g/L yeast extract
5.0 g/L peptone
(nitrogen source)
5.0 g/L sodium chloride
2.0 g/L agar powder
The commercial agar contains the same
composition as the self-made nutrient broth agar. The only difference is that the
commercial agar has 15.0 g/L agar powder instead of 2.0 g/L. For both media,
the final pH should be the same, 7.4.
Preparing culture media manually is useful when small quantities of media are required, while commercial agar is useful when preparing thermally-sensitive media.
As the environment for the
microorganisms, the culture media must be sterilized in order to prevent
contamination that may affect the growth of microorganisms. Autoclaving is a
common method for the sterilization of culture media. This method utilizes
moist heat and high pressure to reach the temperature of 121°C and pressure of
15 psi. The whole process lasts for 15 minutes. Besides culture media, laboratory
instruments and biohazardous waste are also sterilized using an autoclave.
Objective
To prepare sterile nutrient agar for
culturing microorganisms.
Materials
and Reagents
Electronic balance
Beakers
Measuring cylinders
Scott bottles
Glass rod
Spatula
Agar
Peptone
“Lab-lemco” powder (beef extract)
Sodium chloride, NaCl
Yeast extract
Brain Heart Infusion agar powder (BHI)
Trypticase Soy Agar (TSAYE )
Distilled water
Procedure
A) Preparation of 2
sets of 500ml commercial nutrient agar:
1. 14.00g
of commercial nutrient agar powder and 7.50g of agar are weighed by using an
electronic balance, and then placed into the beaker.
2. By
using a measuring cylinder, 500ml of distilled water is measured. The
substances that just prepared in step 1 are mixed together and dissolved with distilled
water. The mixture must be stirred by using a glass rod until it is mixed well.
3. Then,
the mixture is poured evenly into a labelled Scott bottle that have been sterilized.
4 Steps
1 to 3 are repeated to prepare another set of 500ml of commercial nutrient
broth.
5. The
Scott bottles are loosely recapped and autoclaved at 121°C for 15 minutes as
shown in Figure 6.
6. The
medium are removed after autoclaving. The broth preparations are allowed to
cool and then the cap of each bottles are tightened.
B) Preparation of 2
sets of 500ml self-made nutrient agar:
1. 2.50g of peptone, 1.50g of “Lab-lemco”
powder, 2.50g of NaCl, 1.00g of yeast extract and 1.00g of agar are weighed by
using an electronic balance, and then placed into the beakers respectively.
2. By
using a measuring cylinder, 500ml of distilled water is measured. The
substances that just prepared in step 1 are mixed together and dissolved with
distilled water. The mixture must be stirred by using a glass rod until it is
mixed well.
3. Then,
the misture is poured evenly into a labelled Scott bottle that have been
sterilized.
4. Steps
1 to 3 are repeated to prepare another set of 500ml of self-made nutrient
broth.
5. The
Scott bottles are loosely recapped and autoclaved at 121°C for 15 minutes as
shown in Figure 6.
6. The
medium are removed after autoclaving. The broth preparations are allowed to
cool and then the cap of each bottles are tightened.
C)
Preparation of 100ml of Brain Heart Infusion agar (BHI):
1. 5.20g
of BHI agar in powder form is weighed by using an electronic balance, and then
placed into the beaker (shown in Figure 1).
Figure 1: 5.20g
of BHI agar in powder form is weighed by using an electronic balance.
2. By
using a measuring cylinder, 100ml of distilled water is measured.The nutrient
media that just prepared is mixed up with the distilled water. The solution
must be stirred by using a glass rod until it is mixed well (shown in Figure 2).
Figure
2: The solution is stirred by using a glass rod until it is mixed well.
3. Then,
the solution is poured into labelled Scott bottle that have been sterilized
(shown in Figure 3).
Figure
3: The solution is poured into labelled Scott bottle that have been sterilized.
4. The
Scott bottle is loosely recapped and autoclaved at 121°C for 15 minutes as
shown in Figure 6.
5. The
media is removed after autoclaving. The broth preparation is allowed to cool
and then the cap of each bottle is tightened.
D)
Preparation of 100ml of Trypticase Soy Agar with Yeast Extract (TSAYE):
1. 4.00g
of TSAYE agar in powder form is weighed by using an electronic balance, and
then placed into the beaker (shown in Figure 4).
Figure
4: 4.00g of TSAYE agar in powder form is weighed by using an electronic balance.
2. By
using a measuring cylinder, 100ml of distilled water is measured. The nutrient
media that just prepared is mixed up with the distilled water. The solution
must be stirred by using a glass rod until it is mixed well.
3. Then,
the solution is poured into labelled Scott bottle that have been sterilized.
4. The
Scott bottle is loosely recapped and autoclaved at 121°C for 15 minutes as
shown in Figure 6.
5. The
media is removed after autoclaving. The broth preparation is allowed to cool
and then the cap of each bottle is tightened.
Figure
5: Preparation of culture medium.
Figure 6: Scott bottles are placed into the
autoclave chamber.
Results
4 types of culture media have been prepared : 1000ml of commercial nutrient agar, 1000ml of self-made nutrient agar, 100ml of Brain Heart Infusion agar (BHI) and 100ml of Trypticase Soy Agar with Yeast Extract (TSAYE).
Discussions
Nutrients are important to support the
growth of microorganisms. So, cultivation of microorganisms required
preparation of culture media which contains nutrients needed by microorganisms
such as carbon and nitrogen sources. Agar can be easily distinguished from other
materials commonly found in laboratory due to its distinctive smell. Chemically,
agar is a polymer made up of subunits of the sugar galactose, and is a
component of the cell walls of several species of red algae that are usually
harvested in eastern Asia and California. Laboratory agar looks gelatinous when
dissolved in boiling water and cooled. Agar is used for culturing bacteria
instead of gelatin because it won't be degraded (eaten) by bacteria. Also, agar
is firmer and stronger than gelatin. The most common growth media for
microorganisms are nutrient broths and agar plates.
Preparing culture media manually can be labour intensive and very time-consuming, but appropriate amount of media could be prepared without excess waste. Although commercial culture media offers a time-saving solution, it does not address the need to accurately predict the quantities required for future testing.
Sterilization
is important to protect ourselves and prevent contamination. Reliable
sterilization with moist heat requires temperatures above that of boiling
water. These high temperatures are most commonly achieved by steam under
pressure in an autoclave. Autoclaving is the preferred method of sterilization,
unless the material to be sterilized can be damaged by heat or moisture.
There
are several precautions that we need to take during the experiment. Firstly, we
must read the label and instruction on the container before use during
preparation of commercial media, BHI media and TSAYE media. In the progress of
experiment, use distilled water to clean all the apparatus. Measuring cylinder
is used to measure the volume of distilled water required accurately.All the Scott bottles must be labelled first.
The proper receiver for the material must be
selected. The receiver’s weight plus the weight to be measured must not exceed
the maximum load for the balance. The size and shape of the receiver should
permit it to fit into the space and on the balance pan without interfering with
any operation. It is important that the receiver is clean and in dry condition.
Make sure the surrounding of the pan and the pan of the balance is clean. Place
the receiver on the center of the pan of the balance. Then, press the
appropriate tare key every time after the receiver with substances is put onto
the balance to avoid zero error on the balance. Carefully add the powdered
material using a spatula until the desired amount is added. Handle with care to
avoid spilling.If solids are spilled, remove the receiver and sweep out all the
spilled material from the balance using a brush.The spilled material must be properly disposed.
When
dissolving the agar powder, the distilled water in the measuring cylinder
should not be poured all out into the beaker, because there should be some
distilled water reserved for the washing of leftover powders from the weighing
container into the beaker. This step is to ensure that all the agar powder has
dissolved in the culture medium.The media should be stirred evenly by spatula
before pouring into the Scott bottles to ensure the powders mix well with
distilled water.
Before autoclaving, the caps of the Scott
bottles should be loosened. This is because the autoclave machine operates
under high steam pressure and by loosening the cap will allow expansion of the
bottle so that the bottles will not be broken.The autoclaving machine should be
closed tightly so that the media can be sterilized at 121 °C.The temperature is checked so it is
always maintained at 121 oC and the pressure is ensured to
reach 103 kPa above the atmospheric pressure, with steam is continuously forced
into the chamber.The time for destruction of the most resistant bacterial spore
is now reduced to about 15 minutes. For denser objects, up to 30 minutes of
exposure may be required. Then, the exhaust valve is opened to ensure the
pressure drops to nearly 0 kPa before removing the basket with Scott bottles
from the autoclave chamber.
However, after the autoclaving process has
ended, the Scott bottles can be removed from the machine and the cap of the
bottles should be tightened. The media are cooled down. The bottles should also
be turned over again for a few times to allow a balance distribution of agar in
the bottle so that no agar will solidify at the bottom of the bottle. This step
is to make sure that the culture agar can be used for the pour-plate in the
next laboratory work.
Conclusion
Throughout
the experiment, the preparation of culture media is important for cultivation
of microorganisms while sterilization of culture media is important for
prevention of other microorganisms’ contamination.
References
5. http://www.microrao.com/micronotes/pg/culture_media.pdf
6.
http://labreport102.blogspot.my/2014/11/introduction-growthmedium-or-culture_24.html
1. No section for Results?
ReplyDelete3. Discussion and Introduction must emphasize on the difference between commercial media and manually prepared media. Why is there a need to learn on manually preparing media?