I've noticed whilst measuring intracellular pH changes under various conditions that when bicarb/gassed buffers sit in the tubes, there is an initial artifactual pH rise in the cells until the solution that had remained in the tubing is refreshed with freshly-gassed buffer, upon which pH returns to normal. After a while, however, the tubing seems to get charged with CO2 and the problem is lessened. I think, therefore, that Tygon may be permeable or absorptive of CO2, which removes CO2 from the solution and consequently elevates the pH. Is anyone aware of a way around this?
Hi Jacob,
In my PhD I worked on long-term (week) imaging of primary cortical cultures. We used slow perfusion (~1-2ml/day, the culture dish contained ~2.5ml total media) through silicon tubing (which is probably even more permissive to CO2 but much more flexible than Tygon). The pH was controlled inside the dish (and if really needed in the media input Falcon tube) by constant pumping of 95% air/5% CO2. That keeps the pH correct (we used media with phenol-red to visualize it) and creates a positive pleasure inside the dish which helps to keep it sterile. Not sure that answers your question but if needed you can read the details in my paper and in references within.
BTW - the problem with Tygon is that it looses its flexibility and grip at the joints and even if Tygon itself is not permeable to CO2 the gas enters from these micro openings. That's why we switched all of our tubing to silicon both in perfusion and general CO2 tanks in the lab.
Hope this helps.
Roman
Hi Jacob. Good news. Cole-Palmer instrument company has a tubing selection guide chart that lists CO2, H2, N2 and O2 permeability of numerous kinds of tubing, as well as acid/base, ozone and UV compatibility. The link is here: https://www.coleparmer.com/tech-article/tubing-selection-guide
One thing to watch out for is chlorides and other contaminants in plastic tubing, which is a problem with many types of tubing. This is why many ultra-high purity industrial applications (such as nuclear power plants) use Tygon. Tygon leaches close to zero contaminants from the plastic to your medium, or so they claim.
Looks like Tygon makes several kinds of tubing, some of which are CO2 impermeable. So I believe you are in business.
Also, maybe a Tygon or Cole-Palmer engineer can recommend special fittings to eliminate all perfusion at points of connection.
Hope this helps.
Best wishes for the success of your project.
Michael--thanks very much for the pointer. It's very helpful, and I see that "plain" Tygon has a permeability of 745 (units?) whereas FEP-lined tygon has a permeability of 5.9, so about 100-fold better. The Cole-Parmer engineer recommended c-flex ultra or PharMed BPT, but the specs for the latter look worse than regular Tygon.
It seems that there is quite a science to tubing selection!
Thanks again for the helpful info,
Jacob
My problem is to give more oxygene to tissues (muscles, brain) of my patients with an Ehlers-Danlos disease and a very great fatigue.
Best regards
You are welcome. Getting high purity tubing that does not create adverse chemical reactions can be challenging. In the manufacture of tubing, there are plasticizers, aromatic hydrocarbons, ions such as chlorides and other impurities, all of which can leach into your medium and interfere with your processes. I'm glad to be able to help. Yes, it is quite a science in itself to select the right components for a project.
Update: I bought some FEP-lined Tygon, and it seems to have largely cured the problem. Who would have thought? Thanks for all of your help!
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