Binder CB 160 - Reference Measurements; CO Reference Measuring; Measuring CO 2 Concentration Indirectly Via the Ph of the Cell Medium

CB (E6.1) 07/2017 Page 97/145
16. Reference measurements
Reference measurements of the temperature, CO
2
, and O
2
(chamber with O
2
control) can be performed
via the silicone measuring port (N) located on the inner glass door. Reference temperature
measurements always take place under equilibrated conditions with both doors closed.
16.1 CO
2
reference measuring
There are three possibilities to perform CO
2
test measurements between the recommended annual
maintenance procedures. To test the CO
2
concentration inside an incubator, see chapters 16.1.1 to
16.1.3.
16.1.1 Measuring CO
2
concentration indirectly via the pH of the cell medium
By using the indirect determination of CO
2
concentration via the pH-value of the nutrient, it is possible to
check the CO
2
concentration inside the chamber. This is a simple method to test the correct CO
2
concentration without any special CO
2
measuring equipment. You need only use an accurate pH indicator
or a pH-measuring electrode, which are standard equipment in cell culture laboratories.
This method is based on the acid base equilibrium of the buffer system in the culture media. NaHCO
3
buffers the common media. From the pH value of the medium, it is possible to conclude its CO
2
concentration. Figure 25 shows the relationship between CO
2
concentration in vol.-% and the pH of
different NaHCO
3
buffered media.
These test systems are not suitable for calibrating the BINDER sensor system.
Recommended procedure:
• Incubate an empty sample with medium for 1/2 day under the same conditions as the cells. You can
perform the incubation in a cell culture cylinder or in a 50 ml Falcon tube with open lid.
• After gassing, remove the empty sample from the incubator and within 5 minutes measure the pH-
value with a glass electrode.
During the measurement, the medium should have the least possible surface contact with the ambient
air, so that the CO
2
can evaporate only slightly. A significant downward movement will happen only
after 5 minutes, permitting sufficient time for measurement.
• In addition, you can of course use pH-test strips (pH range 6 to 8, not bleeding).
4
6
8
10
12
14
16
18
2
3,70 g/l NaHCO
3
2,20
g/l NaHCO
3
0,85 g/l NaHCO
3
6.8 7.0
7.2
7.4
7.6
Value pH
Vo l.% C O 2
0,35 g/l NaHCO
3
1,20 g/l NaHCO
3
Trade names of common media:
DMEM
BME
MEM
Medium 199
Mc Coy
F10
F12
NaHCO
3
[g/l]
3.70
2.20
2.20
2.20
2.20
1.20
1.20
Figure 25: Value pH of NaHCO
3
buffered media as a function of the CO
2
concentration:
Example: If a pH of 7.2 is measured in a medium buffered with 2.20 g NaHC0
3
per liter, there must be
8 vol.-% CO
2
surrounding this medium.