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« It Was The Worst of Times, It Was the Best of Times | Main | Talmudic Question #55 »

November 09, 2009

The Limitations of LB Medium

by Hiroshi Nikaido

LB medium, also known incorrectly as Luria-Bertani medium, is widely used to grow bacterial cultures, mainly because it is easy to prepare and provides a broad base of nutrients. LB broth contains, per ml, 10 mg tryptone (a mixture of peptides formed by the digestion of casein with the pancreatic enzyme, trypsin), 5 mg yeast extract (an autolysate of yeast cells), and 5 or 10 mg NaCl. It was formulated by Giuseppe Bertani in 1951 for studying lysogeny in Escherichia coli. He called it “Lysogeny Broth,” or LB. Notably, the original formulation included 1 mg/ml glucose, which has been dropped in more recent times. This medium was designed for work at low bacterial densities, a key point of this article.

While this may appear a tasty dish for many bacteria of research interest, it is an inappropriate choice for physiological studies wherein reproducibility is required. Since only bacterial cultures in balanced growth (achieved by sufficient time in exponential growth) have a reproducible average cell size and chemical composition, none of the components of liquid media should become exhausted during growth of the culture. Is this the case with LB broth? To answer this question, we must know what limits bacterial growth in LB broth.

Growth of E. coli usually stops, even in the presence of the large total concentration of organic nutrients in LB broth, when the OD600 reaches around 2, corresponding to about 0.6 mg of E. coli (dry weight) per ml. The reason is not difficult to find: LB medium provides only a scant amount of carbohydrates, and surprisingly small amounts of other utilizable carbon sources. Tryptone and yeast extract are mostly composed of peptides of varying length. In their definitive 1968 study of Bacto Neopeptone using gel filtration, Payne and Gilvarg found that there was a clear size limit for the usable peptides at about 650 daltons—which corresponds to the exclusion limit of porin channels determined several years later. The smaller, usable peptides were a minority, perhaps a quarter of the entire mixture. Free amino acids were an even smaller minority, approximately 1% or less of the entire preparation. If we assume a similar size distribution for the peptides in tryptone and yeast extract, we can postulate that the yield of E. coli is limited primarily by the available carbon sources.

Fig1_d'ari

Growth of E. coli MG1655 in Luria-Bertani broth. An
overnight culture was diluted 5,000-fold in Luria-Bertani
broth and cultivated at 37°C with vigorous aeration. (A)
The OD600 (triangles) and cell concentration (crosses)
were measured periodically. (B) The ratio of the OD600 to
the cell concentration was calculated at each point and
multiplied by 109. Source.

In 2007, D'Ari and associates did a careful study of growth of E. coli in LB broth. They showed that very early in the period usually considered to be well within the exponential phase, when the OD600 reached around 0.3, there is an abrupt change in physiology and the cell size begins to decrease. This observation is not new. As acknowledged by the D'Ari group, Wang and Koch in 1978 had similarly shown that, very early in the "exponential" growth phase, E. coli in LB broth goes through a diauxie-like change in growth behavior. D'Ari's group went further and suggested that what matters is not the general availability of amino acids generated by the intracellular hydrolysis of imported peptides, but the availability of amino acids easily utilized as carbon sources. Although these workers monitored only the free amino acids in the medium (whereas the cells were undoubtedly utilizing peptides for the most part), they concluded that during growth in LB broth E. coli cells undergo alterations in carbon nutrition, using the easier-to-utilize amino acids until they were depleted, then switching to the harder-to-use amino acids.

As an aside, I'd like to mention that the conclusion of D'Ari and his associates is attractive to me for sentimental reasons. Back in the 1950s, I was in a laboratory in Japan that studied the sequential utilization of amino acids in complex media. My mentor, Kiyokatsu Kinjo, was immensely impressed by Jacques Monod's study of diauxic growth done under another resource-strained environment—Paris during the WWII—and tried to mimic Monod's work by studying the utilization of amino acids as the sole nitrogen source. Indeed, using a Vibrio strain he showed diauxic growth with various mixtures of amino acids. He established that aspartate and arginine (and later serine and cysteine were also added to this group) are used first, while other amino acids (such as leucine and valine) are not used until the cells enter the second phase.

Although Kinjo was studying the preferential utilization of amino acids as sources of nitrogen, probably their utilization as carbon sources follows a similar pattern since aspartate and serine would generate C4 and C3 compounds that readily enter the central metabolism. We should note, however, that alterations in carbon nutrition occur even in some synthetic (minimal) media. As is well known, E. coli growing in glucose-minimal medium converts glucose into acetate and other organic acids during the first phase; then later in the "exponential growth" phase it grows primarily by oxidizing these organic acids. Thus, changes in carbon nutrition are perhaps an inevitable complication of batch culture techniques.

Are the shifting carbon sources the only cause of alterations in a young E. coli culture growing in LB broth? I would like to propose that another factor may be an even more profound influence: the availability of divalent cations. In the 1980s, Brian Wilkinson noted that the properties of the outer membrane of Paracoccus denitrificans were very different depending on whether the bacteria were grown in a divalent-cation-sufficient synthetic medium or a complex medium containing Bacto-Peptone and yeast extract. He and his student, Sechan Wee, measured the Mg2+ content of these components as 5.4 and 29 µmol/g. (The Ca2+ content was much lower.) This very important paper is not included in the Medline database, and has been cited only twice (once by myself). If we assume that Difco Tryptone has a similar Mg2+ content, the total Mg2+ content of LB will be only about 200 µM.

However, E. coli needs a lot of Mg2+ and Ca2+ to bridge the highly negatively charged LPS molecules in its outer membrane. The total amount of Mg2+ associated with E. coli cells is estimated to be around 100 mM. (This information is accessible by subscription here.) If a liter of LB broth eventually produces 2 g (dry weight) or about 6 ml (cellular volume) of E. coli, we would need 600 μmol of Mg2+, three times more than what is available in LB. The LPS structure can be modified through the PhoPQ system and E. coli undoubtedly survives even under these Mg2+-starvation conditions, but the Mg2+ content of the cells, especially of the outer membrane, changes throughout the "exponential phase" and the cells are not expected to be too happy. The situation would be much worse if we accept the experimentally determined value for the Mg2+ content of LB broth cited by Papp-Wallace and Maguire in the EcoSal chapter (accessible by subscription here): 30-40 µmol per liter. My feeling is that this serious problem with LB broth has been neglected for many years. It may even be that E. coli grown in LB broth often comes to the stationary phase due to the lack of Mg2+! Certainly one cannot blindly use LB medium if one is observing phenomena involving the integrity of the outer membrane or ionic interactions of LPS, such as the susceptibility of bacteria to polycationic drugs (e.g., aminoglycosides or polymyxin). In this sense, clinical microbiologists are ahead of physiologists; for such purposes they use exclusively "cation-adjusted" Mueller-Hinton broth that contains about 500 µmol per liter of both Mg2+ and Ca2+. I also note that the MOPS medium of Neidhardt, perhaps the only rationally-designed minimal medium, contains more than 500 µmol of Mg2+ per liter. I hope that my colleagues will pay more attention to the availability of divalent cations in the culture medium.

Finally, one must point out that LB media sold as premixed capsules by certain companies contain bile salts as a contaminate of the "beef" product present in the medium. The contamination is sufficient to prevent the growth of mutants that lack the multidrug efflux pump (AcrAB-TolC) of E. coli, one function of which is to protect the cells from bile salts in their environment. This unpleasant fact speaks of the differences between batches of premixed medium. Unless made from scratch, LB broth can vary from batch to batch, thus cannot be considered reproducible.

The bottom line is that, from a multitude of perspectives, the use of LB medium is to be discouraged, especially for use in any studies in which the physiological state and metabolic functions of the cell matter. However, I must confess that I have been guilty of committing this sin many times in my career.

Nikaido2




Hiroshi Nakaido is PBD Faculty Scientist, Structural Biology Department, and Professor of Biochemistry and Molecular Biology, UC Berkeley.

Comments

I don't know where this University of Illinois business is coming from maybe Bertani was a faculty member there eventually? I don't know anything about that but anyway, Luria and Bertani were in Indiana University.

Richard D'Ari:
You say that growth "usually stops ... when the OD600 reaches around 2". We routinely find a higher final OD600, between 5 and 7, although the growth rate between 2 and 5+ is very slow.

Isn't it a quite shocking that OD600 is used for so many decades by so many biologists in so many "critical protocols" even though it is entirely dependent on particulars of the spectrophotometer design and thus can vary dramatically from lab to lab?

What is 2 for you might be 5 for me and could, in theory, be 25 for someone else.

I frequently suspect that "OD600" is a single biggest source of irreproducibility in microbiology.

Elio replies: You are dead right. I checked it our myself and found that the results varied with the machine.

Do everybody know of amino acid compound of LB medium? pawel.filipkowski[atat]pg.gda.pl

Luria wasn't an author on Bertani's 1951 paper that intorduced LB medium, but he was Bertani's advisor at the University of Illinois.

Many thanks, Mark. I wish I could say that people were acronym-shy in the old days, but that's probably not the case. But maybe Bertani was.

Elio

Regarding the name "LB", here it is from the man himself (in the postscript):

http://jb.asm.org/cgi/content/full/186/3/595?view=long&pmid=14729683

When members of my lab in Paris, in the 1970s, were
studying various thermosensitive E.coli strains carrying mutations in
genes of DNA synthesis (many in dnaB and some in other genes) we
(including Dick D'Ari) found that the thermosensitivity of several of
these strains depended critically on whether LB was made up with 5g/l or
10g/l NaCl.

I also found the D'Ari article intriguing. Enjoyed the interesting and lucid further discussion here.

Seems like a logical extension would be some discussion of LB alternatives: what media might we standardize on for general-purpose bacterial growth at a good rate that are suitable for meaningful physiological studies? Are the broths commonly used for plasmid preps or in cloning, such as Terrific Broth or SOC, more desirable? Certainly, they support additional growth and include divalent cations and carbohydrates, but perhaps the emphasis on rapid growth would suggest these also aren't the best choices. Other ideas?

Elio replies:
I was afraid of that! It's easy to knock L broth but not so easy to come up with a substitute. In my view, SOC is more desirable than Terrific, being that SOC contains minerals and glucose. It's merit has been to increase transformation efficiency in E. coli. I don't know if sufficient physiological measurements have been made to warrant recommending it for general use. Any ideas about how to reach consensus?

Another interesting point is the source of LB components. I realize few years ago that depending on the Triptone and/or Yeast extract used in the plates we observed great differences in siderophore formation in Pseudomonas.
This fact support the inappropriate choice of LB medium for studies wherein reproducibility is required.

Elio replies:
Thank you for adding yet another reason why this medium should be avoided in physiological research.

Richard D'Ari writes:

Thank you for your interesting article on LB broth. Let’s hope that it’s heeded by those studying bacterial physiology! I have a few minor comments.

(1) You point out that Bertani’s 1951 medium, the ancestor of today’s LB broth, differed from present recipes in that it contained 1 mg/ml glucose. Another difference is that many recipes used today call for the addition of 1 mM CaCl2 after autoclaving. This is relevant with respect to your concern that LB broth may be limiting in divalent cations.

(2) You say that growth "usually stops ... when the OD600 reaches around 2". We routinely find a higher final OD600, between 5 and 7, although the growth rate between 2 and 5+ is very slow. This slow period nevertheless triples the growth yield, so it’s a true growth phase.

(3) We found that if one continues cultivating the cells beyond the maximum OD600, the OD actually drops. Typically, if a culture reached an OD600 of 7 after 48 hours, its OD then dropped reproducibly to around 5 in the next day or two. There was no detectable lysis and no decrease in protein/ml. This drop in OD600 may reflect a reduction in cell size (for a given level of protein/ml, smaller cells are known to scatter less light than larger cells).

Thank you to Hiroshi for drawing attention to our work on divalent cations and the structure function of the Paracoccus denitrificans outer membrane. This was pure curiosity driven research done back in the day when this seemed a perfectly reasonable thing to do. I never applied for a grant to work on this organism. I was first introduced to Paracoccus in Dave White's lab at the University of Kentucky in the early 70s when Lucille Smith brought it to the lab. The bacterium attracted notoriety because its electron transport chain was the most similar to that of the mitochondrion.

Very interesting article! LB was a source of confusion to me during my research years. Depending on who I asked about it (or which book/journal I read), LB was either translated as "Luria broth" or "Luria-Bertani". But the composition was the big problem: different recipes for LB were even used in the same lab without anybody noticing (or caring about it)! It's true that most people only used LB in that lab for producing plasmid DNA in E. coli, but it was sporadically used for other purposes and other bacteria... So much for reproducibility!

On an aside note:

Congratulations to Elio and Merry, and all the guest bloggers of Small Things Considered, for the non-profit public relations award from PR News for best blog!

A news release can be found at EurekAlert!:
http://www.eurekalert.org/pub_releases/2009-11/asfm-stc111109.php

List of winners at PR News Online:
http://www.prnewsonline.com/awards/nonprofit2009-winners.html

In regards to the requirement for Ca++ to stabilize the outer membrane, there was a short note in the Journal of Bacteriology (179:3350–3353) several years ago that highlights the requirement for Ca++ to retain normal cellular morphology in Deinococci. The article shows through microscopy the consequences of a medium with insufficient calcium, or when the calcium is chelated with EGTA, so the requirement for specific divalent cations is widespread phylogenetically as well!

For a microbiologist edging ever closer to middle age, I think this is a very useful article (the part about glucose in the original formulation in particular). I only use LB for molecular work, because messing with the medium changes the antibiotic sensitivity. Possibly a topic for another post? If you are working with environmental bugs, a teeny tiny amount of Yeast Extract will make lots of things grow, and when you see how they grow, you will wonder why you added all that extra stuff! Lots of stuff (esp fungi, but some bacteria too) will grow on water agar too (or pbs agar)! A real eye opener for students...

Thank you for this nice article! It is eye opening article for young researchers like me.

Elio replies: Glad to hear that you feel that way. It should be an even greater eye-opener for some seasoned researchers who insist on using LB just because it's what others before them have done. Let us hope that younger investigators will avoid the mistakes of their elders.

Can you provide the references for "He established that aspartate and arginine (and later serine and cysteine were also added to this group) are used first, while other amino acids (such as leucine and valine) are not used until the cells enter the second phase."? I'm a graduate student who is also interested in growth on amino acids as nitrogen sources. Thanks!

Interesting that you mention that besides the limitations of the broth itself, there is the misconception about the name. I was a grad student in the same department in Urbana as Bertani was (although forty some years later), and we called it just "Luria Broth" -- and Luria wasn't even an author on the paper which introduced the broth!

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