The best way is to use a chemostat, a small 1 liter fermenter that adds NaOH when the pH decrease below the fixed limit you have previously adjusted

If you have access to a pH meter, you can measure the pH as you add HCl or NaOH solutions drop by drop. But most LB doesn't require any pH adjustments, unless you're dealing with a really fastidious organism

Usually, pH of LB solution is a little bit below 7, and ideally it should be around 7-7.5. I use NaOH added drop by drop to bring the pH to the desied value.

Best of luck.

Generally the pH decreases during bacterial growth. In my opinion, to study the growth at different pH values​​, you should adjust the pH of LB broth at the beginning of the experiment with a buffer solution to each of the desired pH. After, inoculate the bacteria and incubate

Alternatively, you can grow the E. coli in M9 Minimal Medium, that is a phosphate buffer plus glucose and ammonium chloride as the unique carbon and nitrogen sources. The pH of the phosphate could be buffering in a quite wide region from 5 to 7.8 for cell culture. The better if you can access a high density fermenter which equipping with pH probe and feeding pump for HCl and NaOH solutions. That definitely could be control the pH in +/- 0.2 region

Even in microbiology the rules of chemistry are valid and buffering a solution is pure chemistry. Their are two rules of tumb you have to follow when buffering a solution. First of all you have to choose the right type of buffer for the right range you want to buffer, and secondly you have to have a buffering capacity which is high enough tomaintain the pH during the cultivating period. For the buffering range, you have to pick a salt with a good pKa value. This means a pKa which is not more than one unit away from the pH you require. It is impossible to buffer a solution at pH 3.0 with a buffer like MOPS which has a pKa=7.31. MOPS is ideal to buffer a medium in the range of pH 6.31 and 8.31. Once you have chosen the right buffer salt you need enough buffering capacity. In other words you need enough molarity to counteract the acid or base produced by you bacteria in culture. If your bacteria produce millimolars of acid their is no need for molar buffering capacity. Start with a capacities in the range of 50 mM. If it is to low you will notice it immediately when you measure the pH at the end. Adjust accordingly. Pay attention not to dose to high as very high ionic strenght may become lethal for bactria due to osmotic shock. Some examples of pKa's of good biological buffers are: MOPS=7.31, HEPES=7.66, TRIS=8.06, Ethanolamine=9.5 and CAPS=10.51.

Of course there are also some pitfalls with buffers. Some biological buffers can be used as carbon, nitrogen or phosphor source. So be aware that your buffer can be used by your bacteria in an unexpected way. It is also possible that some components of your medium will precipitate if the pH is alteredto strong. So be aware if you notice precipitation when preparing your medium and don't mistake it with turbidity.

Yes, generally the pH decreases during bacterial growth, and biological buffers can be used as carbon, nitrogen or phosphor source. Moreover, the culture must be mantained sterile. To make a culture in a flask is not worthy, you should use a small fermenter, that allows for constant pH maintenance

Patrick,

I totally agree that there is a difference in pKa's at different temperatures. The difference for me is smaller as I normally work with soil bacteria which are killed at 37°C. However, the difference in pKa value s is very limited and only of significance for analytical chemists. For microbiologists and certainly for bacteria a difference of 0.3 pH units is not a big deal and often the accuracy of the way pH is measured in complex media is lower than 0.3

As long as culture will grow pH will drop due to release of CO2 in respiration and other metabolic process depending upon your culture organism. It is sure you have to add base to maintain the pH of culture medium at particular value. Doing this in shaking flask will be really complicated. However you can try in small fermentors which provide facilities to maintain the pH of solution with temperature and time period.

If you want to do your experiment essentially in flask then i will advice you to do it manually adding different buffers to provide your culture a constant pH for long duration. The choice of buffer must be really hectic however you can do if you will go with two basic points; first it should not be harmful for your culture and second it has strong buffering capacity for your pH range.

The LB medium pH is decreased during bacterial culture growth. It depends on anaerobic or aerobic conditions, carbon source type and amount used , initial pH, growth duration and phase, bacterial species and the other factors. Buffering capacity of the medium is also important, to avoid pH decrease buffer (for instance, potassium or sodium phosphate) would be added. It is possible to adjust medium pH by using small amounts of HCl and NaOH.

I would make an adjusted LB broth with potassium phosphate salts as buffer. That way drastic pH changes is avoided

The pH of LB medium changes when the bacteria starts growing. Its better to adjust the pH when you prepare the culture medium using the buffer solution.

sorry guys I know this is an old question. I am planning to prepare ph adjusted broth also. do I adjust the ph before autoclaving the broth, or after autoclaving the broth? thanks

Phosphate based buffer is the more commonly used and less expensive buffer for high density E. coli cultures. You might consider the following based on the phosphate buffer used for the Terrific Broth formula (see Teknova website, a supplier of bacterial culture media):

Add to the LB proth (or TB broth) 100ml of a sterile 10x solution of 0.17M KH2PO4, 0.72M K2HPO4 (The 10x solution is made by dissolving 2.31g of KH2PO4 and 12.54g of K2HPO4 in 90ml of deionized H2O. After the salts have dissolved, adjust the volume of the solution to 100ml with deionized H2O and sterilized by autoclaving).

The initial pH is probably not critical as long as in the physiological range; as noted above production of acetate with growth acidifies the media, which is what needs to be buffered. Also usual phosphate concentrations present in the usual media may be growth limiting as is magnesium (see the seminal paper by FW Studier: Protein production by auto-induction in high-density shaking cultures. Protein Expression and Purification 41 (2005) 207–234, available online).

Dear Scientists, I need to see the effect of pH on the bioactivity of the bacterial culture supernatant, I need to test supernatant samples after pH adjustments to 5.0, 6.0, 7.0 and 8.0 values, and to use the pH-adjusted samples for bioassays. May You kindly answer which buffering agent shall I use in such a range of pH values? and How? the active ingredients in the supernatant are mostly proteins and peptides.

I fully agree with the answer of Dr. Schremmer. You can follow their recommendations,

Dear Salim

For buffering the medium you can use morpholine ethane sulfonic acid.

Hello everyone

I am making 2% malt agar plates by adding 7.5g agar and 10g malt extract in 500ml of DI water. I want to bring the pH of the media to 5 and for that, I would be using a buffer of 0.1M sodium acetate and 0 .1M acetic acid diluted to 200ml using dI water. I want to know if I should make the agar media first say 300ml and then pour the buffer, adjust the pH and then autoclave or I should just make the entire media in buffer (500ml), adjust the pH and autoclave..?