I prepared culture media in Na2CO3-NaHCO3 buffer solution in order to adjust the pH of the media to 9. But after 36hrs, there was not any growth in the petries. So I measured the pH of media and I found that the pH is around 7.5 while it was exactly 9 at the time of preparation (after autoclaving and before pouring in petries). I have also measured the pH of the same petri without inoculation and its pH was also 7.5. It seems either buffer or its mixture with ingredients of media doesn't work properly. I was really confused. What is your suggestion?
The media is composed of 20g nutrient agar, 10g ammonium sulfate, 0.0024g nickel chloride per 1 liter of the buffer solution. I followed the recipe from the link below to prepare the buffer solution: http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html
Dear Harmed
I think that the problem is the volatility in some of the buffer components, as well as your choice of buffer. What may be happening is that at pH 9 some of the ammonium is dissociated to ammonia and lost. The acid dissociation constant (pKa) of NH4+ is 9.25. This will mean that protons are released shifting the solution to a more acidic pH. Furthermore the Na2CO3/NaHCO3 equilibrium has a pKa of 10.33 which means that is not a good buffer at pH 9.
I suggest that you use another buffer system and consider replacing the N-source with something like urea or N-rich amino acids like Lys, Gln, Asn or Arg (do not use His with the nickel in the medium). For alternative buffers refer to http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html. You can also try ammonium carbonate as buffer.
Hope it helps.
Regards
Marina
Bare in mind that carbonate easily is removed from solution in the form of carbon dioxide, especially when the solution is warm. Also, many buffers show a change in their pKa values as a function of temperature. So if your pH is 9 after autoclaving, let's say the temperature of your solution is around 60 C or more just before pouring on the dishes, at room temperature or less it's probably going to be different.
try growing on a TBagar plate if you want to maintain pH closely. Remember to put in the filter sterilized sodium phosphate stock solution after autoclaving.
TB agar
1.2% Bacto tryptone, 2.4% yeast extract, 1.6% glycerol, 1.5% agar, 72mM K2HPO4, and 17mM KH2PO4
Dear Arne, the initial pH (some minutes after autoclaving) was 9 even at 30C.
Dear Hamed
Please write, or the prepared culture media was also maintained at 30C in the further period?
If do not (if the temperature was decreased further to about 20-25C), then you have the answer to your question. That would mean that a change in pH occurred as a result of equilibrium saturation of solution at a lower temperature by CO2 (from the air).
Dear Szymanski
Yes the prepared culture media was also maintained at 30C.
Have a look at this article on pH, temperature and CO2 saturation.
http://www.sciencedirect.com/science/article/pii/S0896844613002441
YOU MAY FIND YOU PROBLEM SOLUTION IN
http://www.sciencedirect.com/science/article/pii/S0896844613002441
Bicarbonate buffer is difficult to maintain as one of the main components in the buffer which is CO2 equilibrates with the atmospheric air and the pH does go down. You need to prepare the bicarbonate buffer fresh use within a reasonable time
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