Vox - Low CO2 levels

I checked the CO2 supply to the small stage-top incubator on the Vox today and found that the percentage making it through the CO2 enrichment device to the work-head was less than 2%. I've now bypassed the enrichment device, which is the box in the picture below, and I'm regulating the CO2 flow rate through a needle valve.

Even so the percentage of CO2 actually being produced by the gas mixer for the 5% and 10% lines is currently only 4% and 8%, respectively. I've contacted the company to find out if it's possible to tweak the gas mix to get the correct values. I suspect this will have to be done in the factory. I have adjusted the flow rate so that the 5% line gives 4% and the 10% line gives 8%. I also decided to test the pH of media under the different conditions to find out what effect the lower CO2 levels would have. I only had access to a complete formulation of DMEM, which contained serum, glutamate etc. It contained 3.7 g/L of bicarbonate, which should be used in combination with 10% CO2 to give a pH of 7.4. The nomogram below indicates what pH should be expected with various concentrations of bicarbonate and percentages of CO2.

I didn't have a pH meter probe small enough to fit into the work-head in the Vox chamber so I had to use indicator paper, which was the most accurate thing. The table below summarises the indicated pH under various conditions.

The readings are a bit on the high side compared to the values as read off the nomogram, but I guess indicator paper is not ideal for accurate measurements. Broadly the alkalinity goes up with decreased CO2 concentration, which is to be expected. The one anomaly is the reading from a 10% CO2 incubator, which I expected to be lower. I couldn't easily tell if the paper was reading 7.7 or 7.9 in that circumstance.

Judging from the rough results above, 8% in the Vox chamber looks like it would be reasonable to maintain a suitable pH for most cells in the short term. The only other alternative for those cells that require a closely regulated pH is a 10% CO2 gas mix cylinder linked to a flow rate meter. I actually don't know how many people use 10% and how many use 5%, and I didn't have a medium suitable for 5% (e.g. RPMI) so I couldn't test the pH of this. For critical 5% CO2 applications it might be possible to regulate the 10% line down to 5% by decreasing the flow rate, but this would be difficult and not very accurate.

It's also important to note that this also affects the GFP time-lapses because they run off the same mixer, although they don't have the CO2 enrichment device that caused the trouble for the Vox so they're likely to already be at 4% and 8% (although I'll test this in the week).

My plans so far are as follows:

• The contact the gas mixer company and find out about repairing and also buying a spare/replacement mixer
• To find suitable regulators and flow meters and set up 5% and 10% gas mix cylinders if necessary
• To test the CO2 enrichment device with 5% and 10% CO2 mixes and get it repaired if necessary

How to use CO2 on the Vox

You should just use the taps below to supply CO2 from now on. I've disconnected and removed the CO2 enrichment device to avoid confusion.