LAB
3: PREPARATION AND STERILISATION OF CULTURE MEDIA
Introduction:
A culture medium (growth medium) is a liquid or gel designed to support
the growth of microorganisms or cells.Microbes require nutrients to grow. Different types of cells will grow in
different type of media. There are two major types of growth media which are cell culture and microbiological culture. Cell
culture uses specific cell types derived from plants or animals while microbiological
culture are used for
growing microorganisms, such as bacteria or yeast. The standard solid medium is nutrient agar which is a gelatinous
substance and the basic medium is nutrient broth which is a typically mix of
water, meat extract peptone and sodium chloride. Some fastidious organisms require specialized environments due to
complex nutritional requirements.The broth contains:
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
Autoclaving is a pressure chamber used to steriliseequipment
by destroy the all viable microorganisms at 121 °C/15psi for 15 minutes
depending on the size of the load and the volume of the liquid.Autoclave operates by
using the steam under pressure as the sterilizing agent. High pressure enable
the steam to reach high temperature result in the increasing the heat content
and killing power. Autoclaving 121 °C/15psi for 15 minutesis the thermal death
time(TDT) for most microorganisms (except some sporeformers). Hence, proper
conditions and time are employed, no living organisms will survive.
Autoclaving machine
Objective:
To prepare sterile
nutrient agar for culturing microorgasnisms
Material
and reagents:
·
Beakers
·
Scott bottles
·
Measuring cylinder
·
Spatula
·
Balance
·
Distilled water
·
Commercial
nutrient agar
·
Nutrient broth
Procedure:
The
Commercial Nutrient Broth Agar
1) 13.0000g
of commercial nutrient agar and 15.0000g of agar were weighed.
2) The
broth (with agar) powder was poured into a beaker.
3) The
1000ml of distilled water was measured by using measuring cylinder.
4) The
1000ml of distilled water was then poured into the beaker containing the broth
powder.
5) The
mixture was stirred evenly until the broth powder was dissolved completely.
6) The
solution was poured into two 500ml of Scott bottles.
7) The
Scott bottles were labelled.
8) The Scott bottles were recapped loosely and
was set aside for autoclaving at 121°C for 15 minutes.
9) After
autoclaving, the media was removed. The broth preparation was cooled. The cap
of each bottle was tighten.
The Own
Prepared Nutrient Agar
1) The
5.0000g/L of peptone, 1.5000g/L of beef extract, 5.0000g/L of NaCl, 1.5000g/L
of yeast extract and 15.0000g/L of agar were weighed.
2) The
peptone, beef extract, NaCl, yeast extract and agar were poured into a beaker.
3) The 1000ml
of distilled water was measured by using measuring cylinder.
4) The
1000ml of distilled water was then poured into the beaker containing the peptone,
beef extract, NaCl, yeast extract and agar.
5) The
mixture was stirred evenly until it was dissolved completely.
6) The
solution was poured into two 500ml of Scott bottles.
7) The
Scott bottles were labelled.
8) The Scott bottles were recapped loosely and were
set aside for autoclaving at 121°C for 15 minutes.
9) After
autoclaving, the media was removed. The broth preparation was cooled. The cap
of each bottle was tighten.
Lactobacillus
MRS Broth
1) 13.7875g of Lactobacillus MRS broth
powder was weighed.
2) The broth powder was poured into beaker.
3) The
250ml of distilled water was measured by using measuring cylinder.
4) The
250ml of distilled water was then poured into the beaker containing the broth
powder.
5) The
mixture was stirred evenly until the broth powder was dissolved completely.
6) The
solution was poured into a 250ml Scott bottle.
7) The
Scott bottle was labelled.
8) The
Scott bottle was recapped loosely and was set aside for autoclaving at 121°C
for 15 minutes.
9) After
autoclaving, the media was removed. The broth preparation was cooled. The cap
of each bottle was tighten.
Brain Heart Infusion Agar (BHI)
1) 5.2000g of Brain Heart Infusion Agar
was weighed.
2) The broth powder was poured into
beaker.
3)
The 100ml of distilled water was measured
by using measuring cylinder.
4)
The 100ml of distilled water was then
poured into the beaker containing the broth powder.
5)
The mixture was stirred evenly until the
broth powder was dissolved completely.
6)
The solution was poured into a 100ml Scott
bottle.
7)
The Scott bottle was labelled.
8)
The Scott bottle was recapped loosely and
was set aside for autoclaving at 121°C for 15 minutes.
9)
After autoclaving, the media was removed.
The broth preparation was cooled. The cap of each bottle was tighten.
Trypticase Soy Agar (TSAYE)
1) 4.0000g of Trypticase Soy Agar (TSAYE)was weighed.
2) The broth powder was poured into
beaker.
3) The
100ml of distilled water was measured by using measuring cylinder.
4) The
100ml of distilled water was then poured into the beaker containing the broth
powder.
5) The
mixture was stirred evenly until the broth powder was dissolved completely.
6) The
solution was poured into a 100ml Scott bottle.
7) The
Scott bottle was labelled.
8) The
Scott bottle was recapped loosely and was set aside for autoclaving at 121°C
for 15 minutes.
9) After
autoclaving, the media was removed. The broth preparation was cooled. The cap
of each bottle was tighten.
Distilled water
1) The 500 ml of distilled water was
measured by using measuring cylinder.
2) The 500ml of distilled water was
then poured into a 500ml Scott bottle.
3) The Scott bottle was labelled.
4)
The Scott bottle was recapped loosely and
was set aside for autoclaving at 121°C for 15 minutes.
5)
After autoclaving, the Scott bottle was
removed. The distilled water was cooled. The cap of each bottle was tighten.
Result:
For own
recipe culture media, the broth should contains :
5.0g/L peptone
1.5g/L beef extract
5.0g/L sodium
chloride, NaCI
1.5g/L yeast extract
15.0g/L agar powder
Each
particular nutrient medium above is weighed appropriately and then dissolve in
1.0L of distilled water. Mix well.
We prepared
1L of commercial nutrient broth, 250ml of Lactobacillus MRS broth, 100ml of Brain Heart Infusion Agar (BHI),
100ml of Trypticase Soy Agar (TSAYE) and 500ml of distilled water.
Discussion:
The pan and the balance must be cleaned before weigh the culture medium powders. The media have to be stirred to ensure that all powders from cultural medium
were dissolved evenly in the distilled water before pour the media into the Scott
bottles. The Scott
bottles` cap should be recapped loosely before putting into the autoclave machine. This is to prevent
the Scott bottles from breaking during autoclaving process.
Ensure the exhaust of the autoclaving machine is closed to
prevent the escape of pressure. Once the autoclaving process finished, the
exhaust of autoclaving machine must be opened to lower the pressure until the
pressure is same as atmospheric pressure.
However, the Scott bottles are removed from the autoclaving
machine after the autoclaving process ended and make sure that the cap of the
Scott bottle are tightened. The bottles must be turn over for a few times to
enable the the agar in the bottle distribute uniformly so that no agar will be
sedimented at the bottom of the bottle. This to make sure that the culture agar
can be pour in the next laboratory process.
Conclusion:
Process of preparation and sterilization of culture media
must be carried under the decontamination condition to avoid the culture media
contaminated by other microorganisms. Autoclaving is the most efficient way to
conduct sterilization process.
Reference:
Obtained from www.wordpress.com on 19 November 2014
Obtained from www.cabri.org on 19 November 2014
Obtained from bioprotech102.blogspot.com on 19 November 2014
Obtained from ibglabreportgroup22010.blogspot.com on 19 November 2014
