Exercise

4. Culture Media

Titra3on value

C1V 1 = C 2V 2
Vm1 Vm2
C1 = Concentra3on of the 1st 3trant (acid or base)
V1 = Volume of the 1st 3trant needed to adjust the pH of the 1st sample to
desired level
Vm1 = Volume of the 1st sample 3trated

C2 = Concentra3on of the 2nd 3trant (acid or base)
V2 = Volume of the 2nd 3trant needed to adjust the pH of the 2nd sample to
desired level
Vm2 = Volume of the 2nd sample 3trated

pH indicators used
1. Bromocresol green
Below pH 3.8
(yellow)

at pH 3.8
(light green)

Above pH 5.4
(blue)

2. Bromothymol blue
Below pH 6.0
(yellow)

at pH 7.1
(green)

Above pH 7.6
(blue)

pH indicators used
3. Phenolphthalein
Below pH 8.2
(colorless)

at pH 9.0
(pink)

Above pH 10.0
(fuchsia)

Refer to the color of the indicators used to approximate the pH of the

medium.

Culture Media Prepara3on

Approximate volumes used for the following:
1. Slants
4. Petri dish (90 mm)
BIG = 8 ml
15 20 ml per dish
Small = 3 ml

2. Stabs

BIG = 10 ml
Small = 5 ml

3. Broths

BIG = 10 ml
Small = 5 ml

Culture Media Prepara3on

1. Percent basis (% w/v)
Nutrient agar
Peptone
0.5%
=
Beef extract 0.3%
=
Agar

1.5%
=

0.5 g / 100 ml
0.3 g / 100 ml
1.5 g / 100 ml

Culture Media Prepara3on

2. Per liter basis
Nutrient agar
Peptone

Beef extract
Agar


Dis3lled water

5 g

3 g

15 g
1000 ml

Culture Media Prepara3on

3. Combined
Nutrient agar
Peptone

Beef extract
Agar


Dis3lled water

5 g

3 g

1.5 % = 1.5 g / 100 ml = 15 g /1000 ml
1000 ml

Culture Media Prepara3on

Given:
Nutrient agar
Peptone

Beef extract
Agar


Dis3lled water

5 g

3 g

1.5 %
1000 ml

Prepare for 10 agar slants (big) and 10 plates of

nutrient agar.

Culture Media Prepara3on

SoluOon:
10 big slants = 10 * 8 ml/slant = 80 ml
10 plates = 10 * 20 ml/plate = 200 ml
Total volume = 280 ml
Peptone
5 g/1000 ml = 1.4 g in 280 ml

Beef extract
3 g/1000 ml = 0.84 g in 280 ml

Agar
15 g/1000 ml = 4.2 g in 280 ml

DisOlled water
280 ml

Eect of Steriliza3on (121C , 15 min)

1. On the pH of the medium (indicated by the change in
pH of the indicators used)
General trend a^er exposure to 121C , 15 min:
Lowering of pH: 0.2 to 0.5 units

pH 4: ini3ally light green to yellow green

pH 7: ini3ally green to yellow
pH 9: ini3ally pink to colorless (browning due to Maillard-type
reac3on: It results from a chemical reac3on between amino
acids and reducing sugars, usually requiring heat.)

2. On the gelling power of agar

pH 4: agar is hydrolyzed; loses its gelling power

Combina3on of high temp. and very low pH results to
hydrolysis of agar.

Culture Media
Buers
Buers can be added to the medium prior to
steriliza3on to prevent dras3c change in pH.