Bioreactors in Stem Cell Biology (Kursad Turksen)

2.3

Growth Media

Prepare the Luria-Bertani (LB) growth media in broth and agar

forms for E. coli with the following components for 1 L: 10 g

tryptone, 10 g NaCl, 5 g yeast extract and distilled water. Add

agar agar (2%) with these components for agar media (see Note 3).

2.4

Spectro-

photometer

Use in following the cell concentration of E. coli at the wavelength

of 600 nm.

2.5

Chronometer

Measure the time and also obtain the DO concentration values

corresponding to those times.

Methods

3.1

Installation of the

Bioreactor

1. Use the oriﬁce shape of sparger which is responsible in entering

of air bubbles into the bioreactor through air pump (see

Note 4).

2. Locate the Rushton turbine port in the STR (see Note 5).

3. Install the sampling port, the temperature detector and polar-

ographic type of DO probe inside the bioreactor.

4. Autoclave the bioreactor with all its parts placed inside for

sterilization of 20 min in 121 C (see Note 6).

3.2

Preparation of

the Growth Media

1. Prepare agar medium for sustaining the stock culture in petri

dishes or slant tubes. Weigh the components of tryptone, NaCl

and yeast agar plus agar agar according to required volumes and

mix them in the distilled water. Boiled the solution for

homogenization.

2. Prepare the components of broth media for scale up from agar

petri dishes to end inoculation volume of bioreactor. Start with

amounts of 5 mL of tubes and keep going with 10, 20 and

40 mL of volumes, respectively. Weigh the all components

except agar agar and mix them in the distilled water in the

required quantities (see Note 7).

3. Prepare separately the growth medium for bioreactor in 2 L of

volume.

4. Measure the pH values of growth media and adjust the pH to

range between 7.5 and 8 (see Note 8).

5. Autoclave the all growth media and equipment for sterilization

of 20 min in 121 C.

6. For agar medium: Shortly after sterilization, pour the broth

medium in aseptic conditions into sterile petri dishes/tubes

while at medium temperature of averagely 60 C and allow it

to solidify by cooling.

Aysegul Inam et al.