A book called Molecular Biological Methods for Bacillus called for plating Bacillus subtilis cells on LB-agar plates with the following components:

A general-purpose medium containing per litre:

Bacto-tryptone 10 g

Bacto-yeast extract 5 g

NaCl 10 g

1 M NaOH 1.0 ml

Autoclave (151b/in^2, 30 min). For LB agar plates solidify with 1.5% (w/v) agar; for LB overlay use 0.7% agar.

The first three components and their corresponding amounts are the same as what I normally work with, but I usually add distilled water for plating E. coli instead of NaOH. According to Wikipedia's entry on lysogeny broth, NaOH can be added to adjust LB's pH. But the protocol here mentions nothing of that sort. In other protocols of the book, at least it would say "adjust to pH -- with 10 M NaOH" or 1 M NaOH, etc. So if it is not to adjust the pH for the growth of Bacillus subtilis, what other purpose does NaOH do for the bacteria?

Also, I usually autoclave my LB or LB-agar for 2 hours instead of 30 minutes. Would autoclaving at 2 hours instead of 30 minutes affect NaOH?

  • $\begingroup$ The NaOH is to adjust pH. They must have determined that that much NaOH with those ingredients makes a suitable pH, but I think your much safer to titrate. $\endgroup$
    – canadianer
    Sep 1 '15 at 17:44
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    $\begingroup$ Why 2 hrs? Why change the recipe? @canadianer is quite correct, per usual, the only reason to add a small amount of extremely strong base to your media is to adjust the pH. And yes, if the pH of the broth and the plates is crucial for your experiment it would be much wiser to titrate the solution. The distilled water pH will vary, as will the agar and the other ingredients (slightly, from batch to batch). also, I would be concerned that autoclaving for extended periods of time could also affect pH by breaking down the ingredients in the complex mixtures $\endgroup$
    – mdperry
    Sep 1 '15 at 22:51
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    $\begingroup$ ncbi.nlm.nih.gov/pubmed/11700343 $\endgroup$
    – DonJulian
    Dec 30 '15 at 19:23

I think NaOH is added to bring up pH not to get too acidic. I feel autoclaving LB or LB-agar for 2 hours is too long. During autoclaving, some components are gradually degraded.


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