Introduction
A growth
medium, or culture medium is a liquid or agar designed to support the growth of
microorganisms or cells. Today, there are many types of culture media available
commercially but the two major types are agar plates and nutrient broth, used
depending on the type of cell to be cultured.
Nutrient
broth is in liquid form whereas an agar plate is a type of nutrient broth added
with agar powder so that when the suspension cools down, it will coagulate as a
semi-solid medium. The microorganisms will grow on the surface of the agar and
this makes examination work so much easier as the colonies of microorganisms
remain stationery and clearly visible.
The agar
preparation is similar to the preparation of a nutrient broth. The composition
of both types of growth media is as listed below:
Peptone 5.00 g/L
Beef extract 1.50 g/L
Sodium Chloride 5.00
g/L
Yeast extract 1.50 g/L
The
difference between the two is that the agar-nutrient broth has been added with
15.00g/L of agar powder. The nutrient broth and the agar-nutrient broth
contained essential nutrients for the growth of the microorganisms, therefore
it has to undergo the autoclaving process to be sterilized so that there won’t
be any other unwanted microorganisms growing in the media.
Autoclaving, sometimes called steam sterilization, is the
use of pressurized steam to kill infectious agents and denature proteins. This
kind of "wet heat" is considered the most dependable method of
sterilizing laboratory equipment and decontaminating biohazardous waste.
Usually it utilises moist heat and pressure that reach 121°C or 15 psi in 15
minutes to disinfect medical supplies and laboratory equipment, extending their
useful lifespan.
Objective
To prepare
sterile culture media for microorganisms culturing
Materials and reagents
Commercial
Nutrient Agar
Lactobacillus
MRS Broth
Brain Heart
Infusion Broth (BHI)
Trypticase
Soy Broth (TSAYE)
Peptone
powder
Beef
extract powder
Sodium
chloride
Yeast
extract
Balance
Distilled
water
Scott
bottles
Measuring
cylinder
Glass rod
Beakers
Procedure
Commercial
Nutrient Agar
1.)
13.00
g of the commercial nutrient agar and 15.00 g of the agar are weighed using the
balance and are put into the beaker.
2.)
1000
ml of distilled water is measured by measuring cylinder. The nutrient media is
mixed up with the distilled water. By using the glass rod, the solution is
stirred until it mixes well.
3.)
The
solution is then poured into the scott bottle that have been sterilized.
4.)
The
bottle is loosely recapped and is set aside for the sterilization.
5.)
All
media is sterilized at 121°C for 15 minutes.
6.)
The
media is removed after autoclaving. The broth preparation is allowed to cool
and then the cap of each bottle is tighten.
Own
Prepared Nutrient Agar
1.)
5.00
g of peptone, 1.50 g of beef extract,
5.00 g NaCI2, 1.50 g of yeast extract and 15.00 g of the agar are
weighed using the balance and are put into the beaker.
2.)
1000
ml of distilled water is measured by measuring cylinder. The nutrient media is
mixed up with the distilled water. By using the glass rod, the solution is
stirred until it mixes well.
3.)
The
solution is then poured into the Scott bottle that have been sterilized.
4.)
The
bottle is loosely recapped and is set aside for the sterilization.
5.)
All
media is sterilized at 121°C for 15 minutes.
6.)
The
media is removed after autoclaving. The broth preparation is allowed to cool
and then the cap of each bottle is tighten.
Lactobacillus
MRS Broth
1.)
13.79
g of Lactobacillus MRS broth in powder form is weighed using the balance and is
put into the beaker.
2.)
250
ml of distilled water is measured by measuring cylinder. The nutrient media is
mixed up with the distilled water. By using the glass rod, the solution is
stirred until it mixes well.
3.)
The
solution is then poured into the scott bottle that have been sterilized.
4.)
The
bottle is loosely recapped and is set aside for the sterilization.
5.)
The
media is sterilized at 121°C for 15 minutes.
6.)
The
media is removed after autoclaving. The broth preparation is allowed to cool
and then the cap of the bottle is tighten.
Brain
Heart Infusion Agar (BHI)
1.)
5.20
g of BHI agar in powder form is weighed
using the balance and is put into the beaker.
2.)
100
ml of distilled water is measured by measuring cylinder. The nutrient media is
mixed up with the distilled water. By using the glass rod, the solution is
stirred until it mixes well.
3.)
The
solution is then poured into the scott bottle that have been sterilized.
4.)
The
bottle is loosely recapped and is set aside for the sterilization.
5.)
The
media is sterilized at 121°C for 15 minutes.
6.)
The
media is removed after autoclaving. The broth preparation is allowed to cool
and then the cap of the bottle is tighten.
Trypticase
Soy Agar (TSAYE)
1.)
4.00
g of TSAYE agar in powder form is
weighed using the balance and is put into the beaker.
2.)
100
ml of distilled water is measured by measuring cylinder. The nutrient media is
mixed up with the distilled water. By using the glass rod, the solution is
stirred until it mixes well.
3.)
The
solution is then poured into the scott bottle that have been sterilized.
4.)
The
bottle is loosely recapped and is set aside for the sterilization.
5.)
The
media is sterilized at 121°C for 15 minutes.
6.)
The
media is removed after autoclaving. The broth preparation is allowed to cool
and then the cap of the bottle is tighten.
Distilled
water
1.)
500
ml of distilled water is measured using measuring cylinder.
2.)
The
distilled water measured is then poured into the scott bottle that have been
sterilized.
3.)
The
bottle is loosely recapped and is set aside for the sterilization.
4.)
The
media is sterilized at 121°C for 15 minutes.
5.)
The
media is removed after autoclaving. The broth preparation is allowed to cool
and then the cap of the bottle is tighten.
Results
- 6 culture media has been prepared which
are 1 L of commercial Nutrient Broth ( with 15 g/ L of Agar ), 1 L of
own-prepared Nutrient Broth Agar (NBA) , 250 mL of Lactobacilli MRS broth,
100 mL of Brain-Heart Infusion (BHI) broth, 100 mL of Trypticase Soy Agar with 0.6% Yeast Extract
(TSAYE) and 500 mL of water (H20).
- For the own-prepared NBA, the recipe is as
stated below:
Peptone 5.00 g/L
Beef
extract 1.50 g/L
Sodium
Chloride 5.00 g/L
Yeast
extract 1.50 g/L
Agar 15.00 g/L
- Each particular nutrient medium above are
weighed appropriately and then dissolve in subsequent amount of distilled
water. Those medium are then mixed well and thoroughly with water.
Discussion
1. There
are a few precautions that need to be highlighted when conducting this lab
work:
·
The
pan and the balance is cleaned with a small brush to ensure more accurate
reading.
·
All
of the doors of the electronic balance are closed before weighing and the
‘tare’ button is pressed every time after the beaker with substances is put
into the balance to avoid zero error.
·
All
of the apparatus are cleaned and rinsed with distilled water before used to
avoid contamination.
·
All
of the media are stirred well with a spatula or rod to ensure balance mixing
with distilled water and to maintain the concentration of the media.
·
The
caps of the Scott bottles are tightened until a slightly tight feeling (but not
over tight) is felt to prevent the Scott bottles from breaking during the
autoclaving process.
2. All of
the Scott bottles which contain the different medium are then placed into a
special basket then the whole basket with all the Scott bottles is put into the
autoclave chamber for sterilization. The steps are as stated below:
- The
drain screen at the bottom of the chamber is checked before using the autoclave.
- Any debris noticed is cleaned up for
efficient heat transfer as steam must flush out of the autoclave
chamber. If the drain screen is blocked with debris, a layer of air
may form at the bottom of the autoclave and prevent proper operation.
- The water level is ensured to be higher
than the bottles in the autoclave.
- The cover of the autoclave chamber is
tightened.
- The exhaust valve is tightened too to
ensure the pressure goes up.
- 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. The conditions must
be carefully controlled or serious problems may occur.
- 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.
Conclusion:
Preparation
and sterilization of culture media are very important to prevent contamination
of the unwanted microorganisms. Besides, different types of agar are needed for
the cultivation of different types of microorganisms. Any of the precaution
steps should be carried out carefully to ensure unwanted errors to occur. Last but not least, autoclaving is a good
sterilization process.
Reference
1.) obtained from http://ibg102labreports.wordpress.com/2013/04/15/lab-3-preparation-and-sterilization-of-culture-media/ on 21/11/14
2.) obtained from http://pasteristem.blogspot.com/2011/03/lab-3-preparation-and-sterilization-of.html on 21/11/14
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