Measuring pH of bacteria

[Gamewizard]

Hi,

 

Not sure if this should be in homework help ?

 

 

I need to test pH levels of an organism, basically to find out at what pH it grows best, where it stops etc. I have decided on a pH range. I intend to use LB broth/BHI as the growth medium for bacteria (in which i will inoculate my bacteria). (My bacteria is from Enterobactericiae, closely related to E.coli)

 

The pH range i am going for is 3.5, 4.5, 5.5, 6.0, 6.5, 7.0, 7.5, 8. I know certain buffers which are capable of maintaining these pH ranges, and that I can also adjust the pH with NaOH and HCl. But I do not know how I will make my media with these specific pH levels using buffers and how I will maintain them (keep them at same pH) because I have read that pH decreases as bacteria grows.

• Citric acid buffer can be used for making pH of 3.5, 4.5, 5.5, 6.0.
• MES buffer solution can be used for making pH of 6.5
• HEPES buffer solution can be used for making pH of 7.0, 7.5, and 8.0
• Adjust pH with NaOH or HCl

Any ideas on how I can carry out this experiment?

[CharonY]

LB is not a terribly good medium to manipulate pH. In addition, the nutrient levels are not very defined making interpretation a bit trickier.

Typically a minimal medium is the best (if more cumbersome) way to conduct theses tests, in which you substitute your buffer system so that you can control pH and buffer capacity. The problem, of course, is that different buffers can affect bacterial growth independent of pH (some may utilize citrate, for instance). What can be used as buffers, depend to some degree on your organism. Some bacteria are sensitive to high phosphate levels, for example. Generally, for a given pH, choose a buffer that is a) not harmful to your bacterium, b) is not utilized as nutrient c) ~ 1 unit within the pka of the buffer system d) has sufficient buffer capacity. The latter depends on whether it is expected that your bacterium produces significant amount of acids, for example.

 

Typical buffers used are: cabonate, MOPS, HEPES, Tris, Ethanolamine, CAPS, ACES, for example

[Gamewizard]

Yes I understand what you mean. I will have to take in account that, however what I dont understand is if I was to make a buffer solution at pH of 3.5 (Citric acid buffer) how will i do that ? and then do I add the prepared buffer at that pH to my liquid media to make the media at 3.5 pH and then measure growth of bacteria from there? and how much bacteria will i be inoculating ? i am trying to do find some papers who have done this or even just simple method from a site etc but i cannot find anything

So for example I have now found out that to make citric acid buffer at pH of 3.5 (mM 50) I need 0.7119% w/v citric acid and 0.3807 %w/v sodium citrate, but how do i calculate this in grams ? and how do i know it is 50mM

 

[CharonY]

The preparation of the buffer depends on the system. Often it is the weak acid (or base) that is titrated with the conjugate base (acid). Others, like Tris are titrated against HCl. You would have to look up for each system you are interested in.

Note that most bacteria have a relative narrow optimum and do not grow on a wide range.

How you set up the assay is entirely up to you and the bacterium. You could measure detailed growth kinetics (i.e. growth curves) which require liquid broth or you could simply estimate colony size on plates. You could measure generation time or you could have yes/no answers.

 

There is no universal test for everything and you would have to read up what makes most sense (i.e. search lit for the bacterium in question and pH resistance, for example).

 

For the last part you have to understand how molarities are calculated and how percentages work. That is rather fundamental and I advise you really to understand that rather than parrot it from somewhere. If you do not grasp the concept you will be lost. Just to provide a hint: 1 gram of solute in 100 ml total volume equals 1 m/v %. I.e. it is the ratio of the weight with volume.

[Gamewizard]

I have had a chat with someone, and they suggested I try adjusting the pH of my LB broth with simple citric acid alone ? do you think that would work ? and if I wanted to bring the pH up do you think it would be okay to use NaOH ? and just keep measuring with pH meter until the broth is at desired pH ?

[CharonY]

You would change pH but you would have no idea what the buffering capacity of your medium is. Again, a typical buffer systems consists of a weak base/acid and its conjugate acid/base.

 

A citrate buffer could work (though it has some issues) and would be a mix of citric acid and e.g. sodium citrate (refer to the Handerson-Hasselbalch equation for details).

 

Just adding a strong base such as NaOH would raise pH, but you have no clue how stable it is once you add bacteria. I think it is one of the cases where some microbiologists cut corners and disregard basic chemical principles.

If your bacteria happens to be E. coli or something related I would recommend looking into M9 medium.