LURIA BERTANI BROTH

Luria-Bertani broth supports Escherichia coli growth to an optical density at 600 nm (OD600) of
7. Surprisingly, however, steady-state growth ceases at an OD600 of 0.3, when the growth rate
slows down and cell mass decreases. Growth stops for lack of a utilizable carbon source. The
carbon sources for E. coli in Luria-Bertani broth are catabolizable amino acids, not sugars. 

The widely used rich medium called Luria-Bertani broth is popular with bacteriologists because it
permits fast growth and good growth yields for many species. The recipe for Luria-Bertani broth
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 min at 120°C. The
tryptone used is a pancreatic digest of casein from cow's milk, and the yeast extract used is an
autodigest of Saccharomyces cerevisiae. Dehydrated Luria-Bertani broth with the above
composition is sold by Difco and called LB Broth, Miller; with 5 g/liter NaCl it is called LB Broth,
Lennox. 

A great deal of work on Escherichia coli physiology has been carried out with cultures in steady-
state growth. One of the principal advantages of this is the extreme reproducibility of the
physiological state of the bacteria; during steady-state growth in a given medium, a given strain
will always achieve the same state. It is reached after a variable lag period and at low cell
density, such that the quantity of nutrients removed from the medium is negligible and the
concentration of excreted compounds does not affect growth. During steady-state growth, by
definition, the following criteria must hold. (i) All intrinsic parameters of the cells remain constant.
Measured as population averages, these include the mean volume, density, and macromolecular
composition of the cells. (ii) All extrinsic parameters increase exponentially with precisely the
same doubling time. These include the optical density of the culture, the number of cells per
milliliter, the amount of protein per milliliter, the amount of RNA per milliliter, the amount of DNA
per milliliter, etc. (iii) The composition, pH, and temperature of the medium all remain constant
within the cells’ detection limits. Cultures in steady state exhibit balanced exponential growth .

It should be noted that the composition of Luria-Bertani broth is not constant. Variations occur
with age (l-tryptophan, for example, is degraded, especially in the light), with time of autoclaving
(affecting the degree of deamidation of l-asparagine and l-glutamine), and from batch to batch.
These variations may account for those cases in which the bioassay gave a higher estimate than
HPLC analysis. 

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. 

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.