In
study of microorganisms, we need to know how the technique to isolate cells
from natural sources and growing them in the laboratory on synthetic media. Thus,
development of synthetic culture media is played important roles in this field.
Microbes require nutrients to grow. These are supplied by either solid or
liquid culture media. Solid media is used for the isolation of bacteria as pure
culture. ‘Agar’ is most commonly used to prepare solid media. Agar is
polysaccharide extract obtained from seaweed.
The broth contains:
1.5g/L
“Lab-lemco” powder (a beef extract)
1.5
g/L yeast extract
5.0
g/L peptone (a nitrogen source)
5.0
g/L sodium chloride
15
g/L agar powder
An
autoclave is a pressure chamber used to sterilize equipment and supplies by
subjecting them to high pressure saturated steam at 121 °C (249°F) for around 15-20 minutes depending on
the size of the load and the contents. Sterilization is the removal all
microorganisms and other pathogens from an object of surface by treating it
with chemicals or subjecting it to high heat or radiation.
Materials
Commercial nutrient agar
Balance
Distilled water
Scott bottles
Procedure:
400 ml Nutrient
Agar
1. The 5.0 g/L of peptone, 1.5 g/L of beef extract,
5.0 g/L of NaCI2, 1.5 g/L of yeast extract and 15.0 g/L of agar were weighed.
2. The peptone, beef extract, NaCI2, yeast extract and agar were poured into a beaker.
3. The 400 ml of distilled water was measured by using
measuring cylinder.
4. The 400 ml of distilled water was then poured into
the beaker containing the peptone, beef extract, NaCI2, yeast extract and agar.
5. The mixture was stirred evenly until it was
dissolved completely.
6. The solution was poured into 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.
400 ml Nutrient
Broth Agar (self made)
1. 11g of commercial nutrient agar and 6g of agar were
weighed.
2. The broth (with agar) powder was poured into a
beaker.
3. The 400ml of distilled water was measured by using measuring
cylinder.
4. The 400ml 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 Scott bottles.
7. The Scott bottles were labeled.
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.
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.
RESULT
1. We were asked to prepare 4 type of culture media.
2. Data of amount of broth dissolved in certain volume
of distilled water is tabulated.
For
400ml self-made agar
1.5g of beef extract + 1.5g of yeast extract + 5.0g of
peptone + 5.0g of sodium chloride + 15.0g of agar powder + 400ml of distilled
water
For
400 ml commercial agar
11.2g commercial nutrient agar powder + 400 ml of
distilled water
For
100 ml BHI
5.2g BHI + 100ml of distilled water
For
100ml TSAYE
4.0g TSAYE + 100ml of distilled water
DISCUSSION
1. Before
using the pan and the balance, the balance should be cleaned and the reading
should become zero before the powders are weighed.
2. The
media which poured into the Scott bottled have to be stirred to make sure all
the powder dissolved inside the distilled water.
3. The
cap of Scott bottle should recapped loosely to prevent it breaking.
4. For
an effective sterilization, the autoclave must not be overcrowded.
CONCLUSION
Autoclave process is a process that effective in
conducting the sterilization process. With correct way of autoclave process, preparation
of nutrient agar for culturing the microorganisms can be done.
REFERENCE
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