Hey!

First, dissolve the LB broth as instructed by the manufacturer in the desired quantity . When dissolved, Calibrate the pH meter using the standard solutions (4, 7 and 10) and measure the pH of the broth. Slowly add 1N hydrochloric acid and stir gently until you reach the desired pH (6.5). After autoclaving, check the pH again to ensure that there were no variations during sterilization. I am available for any questions.

Dear Patricio, thank you very much for the information. Currently I have adjusted the pH using NaH2PO4 and Na2HPO4 buffer as I concerned regarding the ion dynamics. My total study is based on intracellular ion dynamic.Once again thank you , I will definitely try that too.

Thanks for the clarification! That makes perfect sense — using a phosphate buffer is a smart choice when your focus is intracellular ion dynamics, especially to maintain a more stable ionic environment.

I shared the HCl-based protocol just as a general reference, but I totally agree that buffering with NaH₂PO₄ and Na₂HPO₄ provides much better control in sensitive systems like yours.

If you later end up testing the unbuffered version or need to adjust for other conditions, feel free to let me know how it goes — I’d be curious to hear the outcomes!

Good luck with your experiments!

You might consider not using LB but rather using MOPS medium, which has become the standard medium for doing physiological work with E. coli. You will get pH changes in LB upon growth. Or if you don't want to use MOPS medium you could at least buffer your LB with MOPS.

F. C. Neidhardt, P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. J Bacteriol 119(3): 736-747

Thank you Prof. Michael for the thoughtful suggestion and wonderful article. You're absolutely right----LB is not ideal for physiological studies due to its undefined composition and susceptibility to pH shifts during growth. Switching to a defined medium like MOPS not only provides tighter experimental control but also aligns with established best practices, as elegantly demonstrated by Neidhardt, Bloch, and Smith in their seminal 1974 paper. I appreciate the guidance and will incorporate this insight moving forward.

Luria-Bertani (LB) broth (usually prepared with distilled water) is a poor buffer and its pH can drift, especially during bacterial growth. That’s why buffering it with MES or, MOPS is highly recommended if pH stability is important.

Composition:

Tryptone: 10 g/L

Yeast Extract: 5 g/L

NaCl: 10 g/L

Adjust pH at 6.5, +/- 0.2

MES: ~50 mM (optimize with your system)

But, for better control over ionic strength and pH, a defined minimal medium like M9 or Neidhardt's MOPS buffer system is preferable. This medium allows you to control potassium levels and observe efflux-specific changes more clearly than complex media like LB.

NB: Ensure pH stability before and after sterilization.

Thank you Sayak for the detailed information..I appreciate the guidance and will incorporate this.