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How to maintain pH of designed culture media? - Autoclaving, metabolism of cells cause changes in pH (Feb/12/2007 )

Hi everyone!

I would like to know your explanations about this topic: maitaining pH in media for bacterial culture
Every time designing a pH for a medium I find that pH is decreased after autoclaving (1-2 units). Although as far as I known that some chemicals serve as buffers like (sodium/potassium)phosphate, carbonate...when they are supplemented in but this problem still happens. How can I solve this problem?
I have tried to adjust pH of the medium after autoclaving by adding sterilized phosphate buffer or tris-HCl. Is this OK?
If so, how should I do with agar medium?



-Phuc V.N.-

What kind of buffer are you using? What is the concentration of buffer you are using? Sounds like the buffering capasity is very small. pH change of 1 to 2 is extremely large for a buffered solution.

example (sodium phosphate buffer)

72mM Disodium hydrogen phosphate with 17mM Sodium dihydrogen phosphate.

Do note some buffers actually decomposed on exposure to heat and thus unsuitable for autoclaving. (eg MOPS and carbonate based buffers)

And yes you can add filter sterilised buffer to your autoclaved media. However pH adjustment will be difficult. As buffers by their nature take a lot of pertubation before a pH change occurs.

If you intend to grow E coli and not interested in phosphate ions, I would choose a phosphate type buffer. It is both a nutrient and a has a buffering range at the right pH. Buffer and nutrient all rolled in one. Tris-HCl can be used, however maximum e coli growth rate will not be attained. E coli doesn't like Tris and high concentrations will actually inhibit growth. (limiting buffering capacity)

What kind of media are you making? I'm curious. biggrin.gif


Thank you, Perneseblue!

I agree with you in the use of phosphate buffer. I usually apply conventional media for general isolation of bacteria in which buffering reagents such as carbonates, phosphates are included. Moreover, I intend to determine pH extreme for growth of some novel strains. Most of my bacterial collection are halophiles therefore at least 3% NaCl is needed.
Does anyone has different ideas?

PhucV.N. [/size]

-Phuc V.N.-