[JIR0981]

Hey there I'm doing a research project studying marine bacteria and I have to special selective media for my project. The problem is the media needs to be at a specific pH 8.6. The media uses pH indicators Bromothymol Blue and Thymol Blue. The final coloration of the media should be a pretty bright green.

http://antoine.frostburg.edu/chem/senese/101/acidbase/indicators.shtml

The trial batch I made for this media didn't exactly turn out so well by adding drops to try to match a green and the research department doesn't exactly like us using digital pH meters for media since it solidifies fast and gunks up the readers.

So I want to try to calculate exactly or close to 8.6.

The media itself(pH indicators included) after the addition of Di water starts off as a reddish orange color. So that puts it out at around 2.

The solution(media) is around 500 ml and starts off pretty acidic. So I was wondering how much mL of NaOH would I need to raise the pH to 8.6 and have a good green coloration. Its been a long time since I did any titration or pH calculation. Any formulas that would help would be greatly appreciated.

https://catalog.hardydiagnostics.com/cp_prod/content/hugo/TCBSAgar.htm

[Xenonman]

I have no idea how to solve this. Borek suggested in a similar situation to measure the amount of NaOH required, then scale it up to what you need.

I only post to point out things to consider when making the calculations: you'd be adding a lot of NaOH, so the added volume is significant. Also, K_w changes with temperature. I know that, and I know people that know that, but they never seem to consider it when working at a temperature other than standard. Does it ever matter?

[JIR0981]

I noticed that with the trial batch I made because what started off as somewhat green when the media taken off the hot plate after boiling quickly changed color once it solidified into a sort of a brownish olive color that had a slight green tinge to it but it was not the green color I was looking for.

Would it be best for me to start off with straight DI water and try to use NaOH to adjust that to around 8.5 or 8.6 with the pH indicators before adding to the media that way I'll have a starting color at room temperature to judge by? But I'm sure once I had it to the media it will probably make it acidic or throw the pH off again.

[Borek]

The problem is the media needs to be at a specific pH 8.6.

Do I understand correctly that you bought the media from HARDY?

After reading the link you provided I think you should start consulting them how to obtain the correct initial pH.

If I read the description correctly pH changes are indicative of what kind of bacteria are in the solution. If so, you need quite precise control of the initial pH. You know, you need to adjust it with a tiny screwdriver, not a hammer

Close to neutral (and 8.6 is close to neutral) that means using buffers. Trick is, the media itself already contains many buffers - and they are not well defined. Every protein, every weak acid salt is a buffer on its own. That in turns means media has some buffering capacity - and if you have to observe pH changes, you want the buffering capacity to be as low as possible. That rules out adding another buffer.

The only other advice is to use pH meter. Solution is not defined well enough for calculations - you don't know exact composition and exact dissociation constants of all weak acids present. (Actually, even if they were all given, I would not trust calculations too much, but that's another story).