INTRODUCTION
Microbes require
nutrients to grow. Therefore, for
microorganisms culturing, we need to prepare a culture
media to provide them enough nutrients to survive. Culturing
organisms with specific nutritional (or even environmental) needs requires
special purpose media. There are different types of culture media suitable for growing different types of cells,
such as basal, enriched,
selective, differential, transport and storage media. A standard solid culture
media is nutrient agar or agar plates. Agar is polysaccharide extract obtained from
seaweed. Agar is an ideal solidifying agent as it is bacteriologically inert, no influence on bacterial growth, it
remains solid at 37°C and it is transparent. The broth contain:
5.0g/L peptone
1.5g/L beef extract
1.5g/L beef extract
5.0g/L sodium chloride, NaCI
1.5g/L yeast extract
15.0g/L agar powder
Autoclaving is a method used in sterilization process. It is conducted by using of pressurized steam at specific condition: 121°C and 15 psi for 15 minutes, to kill infectious agents and denature protein thus causing thus causing destruction of all types of microorganisms, excluding some sporeformers. The pressurized steam produce a heat called ‘wet heat’ which has large amount of vaporisation heat capacity is sufficient and efficient to destroy microorganisms inside the autoclave chamber. Hence, it is commonly used to sterile laboratory glassware, other equipment and waste, surgical instruments and medical waste.
Autoclaving machine
OBJECTIVE
To prepare sterile nutrient agar for culturing
microorganisms
Materials
and reagents
Commercial nutrient agar
Balance
Distilled water
Scott bottles
Spatula
Brain Heart Infusion Broth agar (BHI)
Trypticase Soy Broth agar (TSAYE)
Beef extract
Yeast extract
Peptone
Sodium chloride
Agar powder
Procedure
Commercial Nutrient Agar Preparation
1. 11.2g of commercial nutrient agar
powder was weighed and poured into a 500ml beaker.
2. 400ml of distilled water was measured
by using 1000ml measuring cylinder and poured into beaker which contained the commercial nutrient agar powder.
3. The mixture was stirred by using
spatula until the powder dissolved completely.
4. The mixture was poured into a
labelled scott bottle.
5. The scott bottle was loosely recapped
and autoclaved at 121°C/15psi for 15 minutes.
6. The media was removed after autoclaving and was
allowed to cool before tighten the cap of the scott bottle.
Self-Made
Nutrient Agar Preparation
1) 6.0g of agar powder, 0.6g of beef
extract, 0.6g of yeast extract, 2.0g of peptone and 2.0g of sodium chloride was
weighed and poured into a 500ml beaker.
2) 400ml of distilled water was measured
by using 1000ml measuring cylinder and poured into beaker which contained the mixture of powders.
3) The mixture was stirred by
using spatula until the powder dissolved completely.
4) The mixture was poured into a
labelled scott bottle.
5) The scott bottle was loosely recapped
and autoclaved at 121°C/15psi for 15 minutes.
6) The media was removed after autoclaving and was
allowed to cool before tighten the cap of the scott bottle.
Brain Heart Infusion Agar (BHI) and Trypticase Soy Agar (TSAYE) Preparation
1. 5.2g of Brain Heart Infusion
Agar (BHI) powder was weighed and
poured into a 250ml beaker.
2. 100ml of distilled water was measured
by using measuring cylinder and poured into beaker which contained the powder.
3. The mixture was stirred by
using spatula until the powder dissolved completely.
4. The mixture was poured into a labelled
scott bottle.
5. The scott bottle was loosely recapped
and autoclaved at 121°C/15psi for 15 minutes.
6. The media was removed after autoclaving and was
allowed to cool before tighten the cap of the scott bottle.
These steps were
repeated by using Trypticase Soy Agar (TSAYE) powder.
Result
The
recipe for preparing the self-made nutrient agar is:
Beef extract 1.5g/L
Yeast extract 1.5g/L
Peptone 5.0g/L
Sodium chloride 5.0g/L
Agar powder 15g/L
For preparing 400ml self made nutrient agar, 0.6g beef extract, 0.6g yeast extract, 2.0g peptone, 2.0g sodium chloride and 6.0g agar powder are mixed with 400ml distilled water.
The others culture media that have been prepared are
400 ml commercial nutrient agar (by mixing 11.2g agar powder with 400ml
distilled water), 100ml BHI agar (by mixing 5.2g agar powder with 100ml
distilled water) and 100ml TSAYE agar (by mixing 4g agar powder with 100ml
distilled water).
Discussion
There are certain precautions that have to be taken
during the experiment, including of:
1. All
the scott bottles have to be labelled first.
2. The
surface of balance must be cleaned before weighing any powder to ensure the
accurate quantity of powder can be obtained.
3. All
scott bottles must rinsed with distilled water and be in dry condition before
using.
4. The
media should be stirred evenly by spatula before pouring into the scott bottles
to ensure the powders mix well with distilled water.
5. Before
autoclaving, the caps of the scott bottles should be loosen to prevent them
from breaking due to the high pressure during autoclaving.
6. The
autoclaving machine has to be closed tightly the the media can be sterilised at
121 °C.
7. After autoclaving,
the media are cooled down and the caps of scott bottles are closed tightly.
Conclusion
In this experiment, we learned the importance of
sterilization of culture media and the process of preparing these culture
media. One of the ways to sterilize the culture media is by autoclaving. Sterilization of culture media is important to
prevent the contamination of other microorganisms.
Reference
http://www.microbeworld.org/careers/tools-of-the-trade/culture-equipment/culture-media
https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/culturing-microorganisms-6/culturing-bacteria-58/culture-media-364-5325/
http://medical-dictionary.thefreedictionary.com/autoclave
