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.
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.







