Not sure how to make HEPES or MOPS buffered media - (Jul/13/2012 )

Hello everyone, this is my first post here. I'm a graduate researcher in environmental engineering, and my project requires quite a bit of microbiological work. Unfortunately, I'm not very familiar with the associated techniques. I need to grow anaerobic metal reducing bacteria on acetate and nitrate, and currently buffer them in a bicarbonate/CO2 system. This works well, except for the fact that the CO2 wreaks havoc in my microfluidic reactors. These reactors are 3 microliters or less in volume, and one single bubble will ruin an experiment. I can sonicate and degas my media before injecting it into my reactor to make it bubble free, but that would alter the pH by the removal of CO2.

So, I'm looking into HEPES and MOPS as alternative buffers because I think they can be used without CO2, or any dissolved gases for that matter. I have HEPES and I have MOPS, but I don't know how to use them. For example, I see papers where people add 10 mM HEPES and get a pH of around 7.2-7.4, which is what I want. Do these people just add HEPES to this concentration, or do they add it and then adjust the pH? If they adjust the pH, what chemicals do they use to do this? So far I'm under the impression that I just need to add the buffers directly to the media at certain concentrations, and I'm ready to go. For example, I never need to add acid or base to my previous bicarbonate and CO2 buffered media. Any help would be very much appreciated because I'm so confused at the moment. Also, any information on MOPS concentration would be useful as well!

People often make a 1M stock of the buffer and dilute it as needed. I'm sure you could make it at 10mM instead. NaOH or KOH are commonly used to adjust the pH of HEPES; this is usually done with 10M or 1M solutions, but you may want something more dilute if you are making a stock of 10mM HEPES. If you need it sterile, filter sterilize and store at 4degrees in the dark.

I'm lazy I just buy it as a solution and dilute as necessary.

I'm actually planning to buy it as a 1M solution: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/reagents/hepes.html. I'm looking at the 100 mL volume and plan to put 10 mL of 1M stock into 1000 mL of media to get a final concentration of 10 mM HEPES. In that case, would I still need to add the NaOH? Or do I just dump the 10 mL into my media and continue making it as usual?

They list the pH at 7.2-7.5. If that is the pH you want, dilute as needed and proceed. This is what I usually do (I make stocks at different pH values rather than adjust the pH of the final media).

If you want to be sure the final pH is where you want it, instead of diluting 10ml into the final 1000ml, add it to ~950 ml and check the pH. If the the pH is correct, add H2O to get 1000ml. If it is off, add 1M NaOH in small increments to get the desired pH, then add H2O to 1000ml total.

That's actually the exact pH range I'd like to be in, so if I can just add it to the media then that's perfect. 7.5 is a little on the high end, but I think this will be the result of having a higher concentration of HEPES in the media. Most people recommend a range of 10-25 mM HEPES. So I'd guess that 10 mM will get me something around pH 7.2, while 25 mM might get me around 7.5 or so. Thanks for the help! Last question: If the pH is too HIGH, do I add HCl to take it down? Every site mentions NaOH or KOH, but no one ever mentions adding acids to bring the pH down. I'm a bit confused by this.

HCl should be fine.