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Vox CO2 - Update

Vox CO2 supply - the problem could be a leaky humidification bottle

I did a bit more investigation of the CO2 supply to the Solent work-head in the Vox environmental chamber to find out why media had been going purply-pink in the chamber and why I had been measuring very low CO2 values with my sensor.

I had reason to believe that directly measuring CO2 would not work (see below) so I decided to measure pH directly as best I could because that is the parameter I am trying to control with the CO2 level. I needed a positive control for pH so I placed a dish of DMEM into a 10% CO2 incubator for about an hour and then measured the pH with indicator paper. I got a pH of about 7.7 (+/- 0.3). I then left this dish out of the incubator in air for an hour and measured the pH to be 8.7 after that time (in fact, when a dish was left out for a day or two the pH went up to about 10). I then put the Solent CO2 enrichment device back into the supply line by removing the manual needle valve from the supply, reconnecting the gas lines to the Solent device and reconnecting the small 125 ml humidification bottle. I put the dish of DMEM into the work head, connected it up, set the flow rate and purge time on the front dial of the enrichment device to the default middle values and supplied 10% (nominal) CO2 gas to the chamber for the recommended 1 hour before checking the colour of the medium and measuring the pH. After 1 hour the medium had turned orangey-red and the pH read 7.7, which is what I saw after it had been in the 10% incubator. It was also the same pH as I measured when supplying gas at a rapid rate through the large humidification bottle. This seemed to indicate that there was no problem with the Solent equipment and possibly no problem with the gas mixer either because the medium pH was measured to be the same as that in a 10% CO2 incubator.

I repeated the test later in the week and this time I found that the pH of the media after 1 hour of being gassed by the CO2 Enrichment Device was 8.3, which was too high and inconsistent with what I had measured earlier in the week. I checked the gas connections throughout the supply by bubbling CO2 through a beaker of water and found that the Luer connector out of the humidification bottle was loose. I replaced the bottle with one of my own where the Luer connector is glued in place and measured the pH again after an hour. This time I found that the medium was the right colour and the pH was 7.7.

So far these results indicate that there is a leak in the original Solent gas humidification bottle that sporadically but frequently results in too little CO2 getting to the work-head. I need to do a few more checks on this before I'm confident that the original connections (with repaired or replaced humidification bottle) can go back in place - and I haven't looked at humidification with the Solent equipment back in place yet - so the large humidification bottle and high gas flow rate can stay for now.

The problem with directly measuring CO2 in the Solent work-head

I have concurrently found that the Anagas meter I use to sample gas from within the chamber and measure CO2 is not suitable for measuring the gas under these conditions.

The meter is designed for measuring gas from large volumes like incubators. It pumps gas out of the chamber at a rate of 120 ml/min but the total work-head volume is only 100 ml and the gas from the Solent supply only goes in at a rate of 2 ml/min, which means the Anagas meter is sucking air into the work-head and lowering the CO2 level as it measures. This explains why the gas concentration is measured correctly when the flow rate is high; at high flow rates the supply is able to replace the gas at the same or a greater rate than the meter sucks it out.

I am currently talking to a company that supplies sensors for CO2, pH and other parameters that can be placed in small chambers and can even be wireless (that is, the work-head lid would not have to be lifted to get the probe in). Up until I can get a solution like this I think I will have to use indicator paper because there's nothing else that will go into such a small volume.

What about GFP1 and GFP2?

It's very likely that the same applies to GFP1 and GFP2, but the gas is supplied to the time-lapses in a slightly different way to the Vox so I will need to get onto both of these machines and measure the pH at different gas flow rates until I find a suitable one that supplies enough gas and humidity but does not cause too much evaporation of the water in the humidification bottle.

What about the gas mixer?

The gas mixer was apparently producing gas at percentages of 4% and 8% instead of 5% and 10%. I'm now not certain whether this error is real because of the problems with the meter described above. Having said that, I was measuring the gas in this case in a larger volume and at a higher flow rate than through the Solent equipment, so the meter should have been more suitable under these conditions. I have a new gas mixer that I can use to compare and if necessary replace the other one while it is repaired. I will have to change over the mixers at a future date when I can book all of the machines in that room because they are all supplied from the mixer and will lose their CO2 supply during the changeover.

Unfortunately Volocity does not automatically calculate the size of your dataset before you start a time-lapse. It also does not warn you if you will fill the hard disc up during the acquisition. It is therefore critically important that you calculate how large your dataset will be before you start and work out whether the hard disc will reach capacity before the time-lapse finishes. It may be that you are acquiring more data than you need and just a few simple changes to the acquisition could reduce the size of the dataset dramatically. I have written a technical not that describes how to calculate the eventual size of your dataset, how to check whether you will fill up the hard disc and how you can reduce the size of the dataset or otherwise offset the chances of time-lapse failure due to exceeding the hard disc capacity. The technical note is available as a PDF below:

Timelapse_Data_Size.pdf

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.

Condition

pH (indicator paper)

TC Incubator with 5% CO2

7.9

TC Incubator with 10% CO2

7.7/7.9*

Vox Chamber with 4% CO2

8.1

Vox Chamber with 8% CO2

7.7

Vox Chamber with no CO2

8.5 (after 45 minutes)

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.

This post demonstrates the blog capability of the UCL Wiki. It's one option for creating a news/discussion forum for light microscopy at the LMCB.

I've pretty much satisfied myself that the layout is basically done. I think I'll start adding content from today.