Laboratory Activity No 2

Preparation of Culture Media

Culture medium is a nutrient material prepared for the growth of microorganisms in the
laboratory. Some bacteria grow well in on just about any culture medium; others require special media
and still others cannot grow on any nonliving medium. A culture medium must meet several criteria.
First, it must contain the right nutrients for the specific microorganism to grow. It must contain sufficient
moisture, properly adjusted pH and a suitable level of oxygen or none at all. The medium must be
initially sterile, that is, it must initially contain no living organisms prior to culture of the microorganism
intended to be cultured.

Objectives:
1. To enable students to prepare simple culture media.
2. To emphasize the basic bacteriological techniques to preserve sterility.
3. To give students a hands-on practice on these techniques.

Materials:
Nutrient agar 39 pairs petri plates 1 pressure cooker and stove grad. cylinder (100 mL)
Nutrient broth 31 test tubes 6 Erlenmeyer flasks ( 250 mL) cotton
Aluminum foil alcohol lamp denatured alcohol

Procedure:
A. Preparation of Nutrient Broth
Using a commercially prepared dehydrated medium, weigh out the amount needed to prepare the
required volume of nutrient broth. Follow the manufacturer’s instruction during dehydration. Stir to
completely dissolve the medium and dispense 7 ml in each test tube, cover with cotton plugs. Place
in test tubes in a wire mesh basket or beaker and cover with aluminum foil and sterilize ant 15 psi for
15 minutes.

B. Preparation of Nutrient Agar

Nutrient agar contains nutrients suitable to subculture a wide range of microorganisms and makes it
an excellent agar media to check on the purity before any biochemical or serological test. It contains
1.5% agar which solidifies the nutrient medium, making it suitable for the cultivation of
microorganisms.

B.1. Agar plates

1. Suspend 28g of nutrient agar powder in 1L of distilled water. Add 3% NaCl or seawater
if source of sample is from seawater.
2. Mix and dissolve them completely in water bath.
3. Sterilize by autoclaving or using pressure cooker at 121°C for 15 minutes.
4. Pour the liquid into sterile petri dish and wait for the medium to solidify. Be sure that you
are preparing the agar in the clean environment to prevent any contamination.

B.2. Agar slants

1. Weigh the exact amount of NA good enough for 220 mL DW, follow the rehydration rate
given by the manufacturer which is 28 g/L of distilled water.
2. Mix and dissolve them completely in water bath.
3. Dispense 7 mL of the melted NA into clean test tubes. Plug with cotton and sterilize at 15
psi for 15 min.

C. Sterilization of glassware
Together with the sterilization of the culture media, the petri dishes and other glassware needed for
culture will be sterilized. First wrap the petri dishes as directed by your instructor and place inside
polyethylene bags (PE bags) or aluminum foil prior to sterilization. The test tubes must be plugged
with cotton first and placed inside PE bags and sterilize at 15 psi (121 oC) for 15 minutes.

NOTE: ALL MANIPULATIONS SHOULD BE DONE ASEPTICALLY

Results and Discussion:
1. What is sterilization? Why is it necessary to sterilize the culture medium?
2. The sterile nutrient agar will be dispensed in sterile petri dishes at 45 oC. Why?
3. Why should we store the agar plates stored in an inverted position?
4. What is aseptic technique? Why should all manipulations be done aseptically?