Five Things You Might Not Know about LB
LB medium is a staple in virtually every lab. It’s commonly used to propagate E. coli, and as such will be used frequently by any lab that does cloning. Chances are, LB broth or plates were one of the first things you learned to make as a newbie in the lab. Here are a few interesting facts you might not know about LB:
- LB broth was first created in the 1950’s by Giuseppe Bertani to optimize the growth of Shigella and plaque formation as part of his research into bacterial lysogeny. Many researchers mistakenly believe that LB stands for Luria-Bertani. However its inventor clarified in his review article “Lysogeny at Mid-Twentieth Century: P1, P2, and Other Experimental Systems,” that the initials were intended to stand for lysogeny broth, not his name and that of his colleague Salvador Luria [1].
- Making your own LB broth from scratch is cheaper than buying premixed formulations from companies. Some of these premixes are known to contain contaminants which can inhibit certain strains of E. coli. By making your own LB, you have full control over its composition and can prevent inter-batch variability [3].
- There are many variations of the recipe for LB broth but the most common is as follows: combine 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 1 liter of distilled water; adjust the pH to 7.0 with 1 N NaOH; and autoclave the mixture for 25 minutes at 120°C [2].
- LB broth supports E. coli growth to an optical density at 600 nm (OD600) of 7. However, steady-state growth ceases at an OD600 of 0.3, when the growth rate slows down and cell mass decreases. Growth stops due to the lack of a utilizable carbon source [2].
- You should make up LB just before autoclaving to prevent bacterial contamination (i.e., no more than a couple of hours). Bur remember, don’t add antibiotics to your LB broth before autoclaving, as the high temperature will inactivate them. Once you have added antibiotics to your LB, store it at 4°C.
References
1. Bertani, G., Lysogeny at Mid-Twentieth Century: P1, P2, and Other Experimental Systems. Journal of Bacteriology, 2004. 186(3): p. 595-600.
2. Sezonov, G., D. Joseleau-Petit, and R. D’Ari, Escherichia coli physiology in Luria-Bertani broth. Journal of Bacteriology, 2007. 189(23): p. 8746-9.
3. Nikaido, H. (2009). The Limitations of LB Medium. Small things considered – The Microbe Blog. ASM. https://schaechter.asmblog.org/schaechter/2009/11/the-limitations-of-lb-medium.html
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Does the pH of LB broth drop after autoclaving? We have found that post-autoclaving the pH of the broth is reduced to ~6.3-6.5. Some references mention pH, but not with regard to post-autoclave.
At the end of the article, the author has mentioned that the LB medium can be stored at 4°C after adding antibiotics to LB medium. But can anyone say for how long the medium (with antibiotics) can be stored?
In my experience, it can last about a month—maybe two on the outside (if it’s not exposed to light).
It depends on the antibiotic. With ampicillin it can be stored 1 month at 4C. Kanamycin is more stable,, and my colleagues leave it at room temperature for months. Carbenicillin is also more stable, but I haven’t tested it. Cb plates at 4C work a year later, I think.
Interesting to see you clarify that LB stands for Lysogeny Broth (which we all know) but then continue to use the phrase LB Broth…!
I understand the perception that the only way to control contamination and save costs is to make LB yourself, but I have to strongly disagree that it’s cheaper to make it yourself. These days, there is a long list of companies that will provide it dry, as a liquid broth or plated for you. Teknova will provide in any of these formats, plus anything is fully customizable to match each cusotmer’s unique application and strain. Additionally, LB is also an obsolete formulation for many applications, and there are better products available today. Fair warning – I am a Teknova employee, but while biased, I had to pass this information along. Thanks!