Hi, I'm making up a few different types of nitrogen-free media, including nitrogen-free malate with bromothymol blue (NFb) and nitrogen-free combined carbon (NFCC) media.
NFb medium composition that I'm following is as follows (according to Baldani 2014, Article The art of isolating nitrogen-fixing bacteria from non-legum...
). NFCC medium composition is similar enough that I'm getting the same issue:The problem I'm getting is that after I make up the media, prior to pH adjusting, I've tested the pH and it's all the way up to 12 or so. I would assume that needing to add KOH to make it 6.5 would mean that the pH would start below 6.5. But that's clearly not the case. And even if I wanted to add HCl to get the pH down to 6.5, it would take way too much HCl. This is a problem for both the NFb and NFCC media - am I doing something wrong here? Thanks
Dear Dan, many thanks for sharing this very interesting technical question with the RG community. In the original literature reference which you provided with your question (good idea!) this particular medum is called the basic medium. After adding 4.5 g of potassium hydroxide (KOH) one should nod be surprised to see that pH rise to 12.5. After all, KOH is a strong base. However, there is a mistake in point 11 in your question. The procedure does not call for "KOH to adjust pH to 6.5". This would make absolutely no sense because addition of more KOH would make the medium even stronger basic. Instead, it is stated in the original protocol (citation): "Dissolve in hot-water bath. Complete to 100 mL by adding distilled water), 1 mL; KOH, 4.5 g; Distilled water to bring the final volume to 1,000 mL and adjust pH to 6.5." This means that in the final step you need to add e.g. some HCl solution to bring the pH back to 6.5. I diggest that you can do this before filling up the medium to 1 liter so that in the end you will have exactly 1,000 mL with a pH of 6.5.
I hope this clarifies the issue. Good luck with your work and best wishes, Frank Edelmann
Dear Dr Dan Naylor . I think if you can use a buffer solution of pH 6.5, this can be a solution. The buffer solution is a solution that resists any change in pH . So pH will remain constant . Therefore if you use buffer of pH 6.5 , the medium pH will remain 6.5 always.
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