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Adjusting pH of RPMI 1640 culture medium for 5% CO 2 using HCl

Please note: this method is recommended only as a last resort, if it is impossible to set up a 10% CO 2 incubator. The intracellular pH is affected more by CO 2 /H 2 CO 3 than by HCl, and better results are obtained that way.


  • Hydrochloric acid, HCl, say 1M. Can assume it is sterile.
  • 1x culture medium (RPMI 1640).
  • Cylinder of compressed 5% CO 2 in air, with pressure reduction valve and outlet hose (plastic tubing), preferably with large 0.22 µm filter, to connect to sterile plugged pipette. Alternatively, cylinder of compressed air only, with hose etc.

1. Make up bottle of medium with glutamine, antibiotics etc (if not already included) but without serum (which would froth during gassing). We recommend adding extra phenol red to RPMI 1640 for easier monitoring of pH. Details under Cell Culture Media.

2. Add HCl at 10 mM (final), e.g. 5 ml of 1M acid in 500 ml of medium.

3. pH initially becomes very low because medium is not yet in equilibrium with the atmosphere. This would be toxic to cells. Restore it to correct pH by gassing the medium thoroughly. In other words, bubble through it 5% CO 2 in air from the cylinder, to equilibrium (indicator colour stops changing). This can take a few minutes. Alernatively, carefully bubble plain compressed air until the phenol red indicator gives an orange colour (not yellow, not red).

If neither option is available, it is possible to adjust the gas in the medium by leaving it overnight in the incubator with the screw top on but loose. But this warming will reduce its storage life.

4. NB, we don't know if 10 mM is right for all manufacturers' versions of RPMI. Incubators may vary a little too. If you are not getting an orange colour (pH 7.0-7.1) at equilibrium in the incubator , try adding a little more or less acid (by say 2-mM steps), until you are.

5. If pH of medium subsequently rises by de-gassing (CO 2 loss, medium goes pink) during storage, restore it by bubbling 5% CO 2 again before use. Even a few hours in alkaline medium is inhibitory for melanocytes. Or re-gas in incubator again. (Obviously you can't use a plain air cylinder to increase the CO 2 content, only to reduce it.) If your medium de-gases repeatedly before it is used up, try distributing it to smaller bottles.

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