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LB Media

LB is the most common media used to grow recombinant Escherichia coli (E. coli). LB media was named by Giuseppe Bertani as “lysogeny broth” in due to the research he was conducting on lysogeny. LB is commonly incorrectly referred to Luria-Bertani media, Luria Broth or Lennox Broth. LB media is also used to culture a variety of other facultative organisms.

Components of LB Media:

One reason LB is commonly used is because it is simple to make, with only a few ingredients, tryptone, yeast extract, and sodium chloride (NaCl). Tryptone, a mixture of peptides generated by digestion of casein with the pancreatic enzyme trypsin, provides nitrogen and carbon. Yeast extract provides vitamins (including B vitamins) and some trace elements. NaCl provides sodium ions for transport and osmotic balance.

E. coli derived from the K-12 strain, one of the most commonly used parental strains of E. coli in use in molecular biology today are deficient in B vitamin production.

Historical Background of LB:

LB is a nutritionally rich medium that has been used since the 1950’s to culture Enterobacteriaceae and for bacteriophage plaque assays. LB permits fast growth with good growth yields for many species. In 1951, Giuseppe Bertani developed LB to optimize plaque formation in a Shigella indicator strain of Enterobacteriaceae. Today, LB media is the most common media for growth of recombinant strains of E. coli.

There are three common formulations of LB; LB Miller, LB Lennox, and LB Luria. All are sometimes referred to as LB, though the LB Miller formulation is the common or more standard formulation of LB. The alternate formulations of LB vary in the NaCl concentration.

Formulations of LB (g/L):

Ingredients % Luria Lennox Miller
Tryptone 1.0% 10 10 10
Yeast Extract 0.5% 5 5 5
Sodium Chloride (NaCl) 0.05, 0.5 or 1.0% 0.5 5 10

The confusion about the name LB is understandable given the history of Bertani, Luria, and Lennox. Giuseppe Bertani was a member of the Luria lab at Indiana University when he formulated LB media. Ed Lennox was also a member of the Luria lab and worked with Bertani on some of the early lysogeny experiments utilizing Shigella. Salvador Luria published a paper in 1955 in which he copied the original formulation of Bertani and LB is sometimes incorrectly attributed to Luria due to his scientific stature, and so can also be incorrectly referred to as Luria Broth.

The original Bertani formulation was 1.0% Bacto tryptone (10.0 g/L), 0.5% yeast extract (5.0 g/L), 1.0% NaCl (10.0 g/L), 0.1% glucose (1.0 g/L) pH adjusted to 7.0 with 1N NaOH. Glucose was added after autoclaving. Over time, the addition of glucose has dropped out of all formulations of LB. The name Miller comes from the formulation in the book Experiments in Molecular Biology by Jeffery Miller, published in 1972, which does not contain glucose.

The original and most common formulation of LB, Miller, contains 1.0% NaCl. In 1955, Ed Lennox was studying mechanisms of DNA synthesis utilizing strains of E. coli sensitive to osmotic stress developed a formulation of LB containing half the salt of the original formulation, i.e. 0.5% NaCl . This formulation is referred to as LB Lennox. Today, LB Lennox is used for cultivation of E. coli when using salt-sensitive antibiotics such as Blasticidin, Puromycin, and Zeocin. A third formulation of LB, containing the least amount of salt, is referred to as LB Luria. This formulation contains 0.05% NaCl. LB Luria is used to isolate marine organisms such as Vibrio cholerae.

 Mechanisms of Growth in LB:

LB media was originally designed for growth of bacteria at low densities. Exponential growth, a period of steady-state growth, is estimated to end when the OD600 (optical density at 600 nm) is between 0.6 and 1.0. It is known that growth of E. coli in LB usually stops when the OD600 reaches approximately 2.0 under normal growth conditions, corresponding to approximately 0.6mg E. coli (dry weight) per ml.

In 2007, D’Ari and colleagues undertook a comprehensive study of growth characteristics in LB, looking specifically at the physiology of E. coli K-12, one of the most common strains utilized in molecular biology today. They demonstrated that K-12 cells grown in the LB Miller with a final OD600 of 0.6 -1.0 are not always in the same physiological state. D’Ari and coworkers confirmed earlier observations of diauxic growth (growth in two phases) in LB and noted that exponential growth stopped at ~OD600 0.3, much earlier than commonly thought.

It is known that E.coli is known have poor growth when the pH exceeds 9.0, and it is not uncommon for LB media to change to a pH close to 9.0. However, when D’Ari and coworkers adjusted the pH of LB there was no effect on the growth curve, whereas, when glucose was added to the media, cultures could grow to OD600 6.49. This suggests that growth of E. coli in LB is carbon limited. As noted, the original formulation of LB by Bertani, contained 0.1% glucose. This research demonstrates that while LB is a good medium for routine growth, it should not to be used for physiological studies where reproducibility and a prolonged period of exponential growth is required.

LB and selection:

There are a range of additives that can be added to LB medium for identification or selection of a population of organisms. Antibiotics are often added to LB medium to select for cells that have been engineered to have resistance to one or more antibiotics.

X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) or Bluo-Gal (halogenated indolyl-β-galactoside) may be added to LB medium for blue-white screening for insertion of sequence into a multiple cloning site (MCS) in a plasmid containing the α fragment of the β-galactosidase gene. To induce expression of genes controlled by the lac promoter, the non-metabolizable lactose analog IPTG (isopropyl-beta-D-thiogalactopyranoside), is added to the medium. In bacteria where there is no sequence inserted into the plasmid MCS colonies will be blue since the X-Gal or Bluo-Gal will be cleaved by β-galactosidase to form 5-bromo-4-chloro-indoxyl. For plasmids where there is an insertion there will not be a functional β-galactosidase and the colonies will remain white.

 References:

1. Bertani, G. 1951. Studies on Lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriology, 62:293.
2. Bertani, G. 2004. Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J Bacteriology, 186:595.
3. Lennox, E.S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190.
4. Luria and Burrous. 1955. Hybridization between Escherichia coli and Shigella. J. Bacteriology 74:461.
5. Maloy, S. 1990. Experimental Techniques in Bacterial Genetics. Jones and Bartlett Publishers, MA.
6. Nandi, Nandy, Mukhopadhyay, Nair, Shimada, Ghose. 2000. Rapid method for species-specific identification of vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW. J Clinical Microbiology. 38:4145.
7. Sezonov, Joseliau-Petit, and D’Ari. 2007. Escherichia coli physiology in luria-bertani broth. J. Bacteriology 189:8746.
8. Neidhardt, Ingraham, and Schaechter. 1990. Physiology of the bacterial cell. Sinauer Associates, Sunderland, MA.
9. Green and Sambrook. 2012. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
10. Nikaido. 2009. The limitations of LB medium. The Microbe Blog.
11. Miller, J, 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York