Lysogeny broth, commonly shortened to LB, is the most commonly used rich medium for bacterial culture, especially for E. coli. It is a solution of tryptone, yeast extract, and NaCl.
The main carbon source is the oligopeptides from the two peptones (protein sources): tryptone and yeast extract. Tryptone is the result of digestion of bovine milk casein with the pancreatic protease trypsin. Yeast extract is the autolysate of yeast, where the yeast proteome from ruptured cells is digested by endogenous proteases. E. coli uses oligopeptide permeases and proteases to import and digest tryptone and yeast extract peptides into amino acids, which are utilized as primary carbon sources in order of the strain's preference (1) . As a result, growth in LB does not acidify the medium as with growth on most sugar/carbohydrate carbon sources; LB is alkalified to pH 9 at saturation (1) at which point exhaustion of a carbon source arrests growth (not the alkaline pH) (1) . Yeast extract is a complex, widely used hydrolysate of yeasts that provides amino acids/peptides, nucleotides, water-soluble metabolic cofactor "B vitamins", some carbohydrates, and other growth enhancers, which can enable growth of diverse microbes with differing nutrient needs. Yeast extract content was found to be the main determining factor for plasmid yield in E. coli, resulting in a higher yeast extract version of LB called LB30 (4,6).
LB has limitations; no conclusions about E. coli physiology ought to be made in LB (2) , but rather in a rationally-designed defined medium, like M9 or MOPS minimal (3) .
For improving low- and high-copy plasmid production efficiency/yield, Shyam Bhakta devised LB 30 and variants based on the literature ( Notes).
Note: The term "LB" is used both for the broth and the agar "lysogeny agar", originally LA, and it is often incorrectly(2,12) expanded to "Luria broth" or "Luria-Bertani medium". The common formulation differs from Bertani(12) and Lennox's(13) broths by the omission of 1 g/L glucose, and only LB-Lennox maintains only 5 g/L NaCl, whereas the most common LB-Miller keeps Bertani's original 10 g/L.
- Combine the following in an appropriate sized flask or bottle:
For broth:
- 25 g/L LB powder
or from separate components:- 10 g/L tryptone
- 5 g/L yeast extract
- 10 g/L NaCl for LB-Miller (common). 5 g/L for LB-Lennox. 0.5 g/L for LB-Luria.
- 25 g/L LB powder
For agar:
- 40 g/L LB agar powder
or from separate components:- 10 g/L tryptone
- 5 g/L yeast extract
- 10 g/L NaCl for LB-Miller (common). 5 g/L for LB-Lennox. 0.5 g/L for LB-Luria.
- 15 g/L agar
- 40 g/L LB agar powder
For LB30: Add an additional 25 g/L yeast extract to either recipe for a final 30 g/L.
- Enhancements:
- MgCl2 (or MgSO4): add 5–10 mM from an autoclaved 1–2 M stock. Improves growth yield and rate, reduces cell stress and protein acetylation.(5,Notes). Prefer Cl salt (Notes).
- Buffer: add 1× M9 salts or TB salts from an autoclaved 5× or 10× stock. Reduces cell stress, purportedly. Alternative: roughly 100 mM final Na/K phosphate buffer.
- Carbohydrate: add pre-autoclaved 5 g/L (0.5% m / V ) glucose or 10 g/L (7.94% V/V ) glycerol after autoclaving. Improves cell yield, reduces cell stress. Glycerol catabolism is less acidifying than glucose. May not improve plasmid yield (4) . Only use in combination with Mg and buffer (5,Notes).
- Water: Milli-Q water or deionized water up to final volume.
- Close and shake to partially combine, or stir with stir bar and remove. Flasks can be closed with parafilm or gloved hand for shaking. Remove stir bar from broth with a magnet.
For agar, adding a stir bar is advised for quicker and uniform cooling prior to pouring, unless using a 50–60° water bath. - Sterilize by autoclaving for 15–20 min, 15 psi on psi on the liquid cycle; 15 min for ≤1 L volume(s) and 20 min for >1 L volume(s).
- Make sure bottle lids are very loose and foil lids aren't tight.
- Steam-injection autoclaves don't need water in the autoclave bin.
- Allow the medium to cool to 50–55°C. Add any antibiotics or other thermosensitive additives, e.g. inducers, only when the vessel's temperature has fallen to 50–55°C. Swirl thoroughly to distribute additives if not using a stir bar.
- Cooling can be sped and made uniform by stirring with a stir bar added prior to autoclaving. Cooling rate can be maximized to ≈10 min for 1 L volumes by immersing the flask/bottle with stir bar in a small autoclave bin filled with water and set over a stir plate, stirring rapidly. Stir bar is essential, or else agar will congeal.
- For agar, monitor temperature closely using a tape thermometer held against the vessel surface over the medium, or an infrared thermometer pointed at a dry vessel surface in contact with the medium.
- Plates ought to be poured soon after medium reaches 50°C to allow time for pouring before agar congeals.
- Immersing the vessel in a 50°C water bath, as made using a "sous vide" immersion circulator clipped to the side of an autoclave bin filled with deionized water, will not maximize the cooling rate of the medium but will allow the most time flexibility when dispensing agar, as the agar will cool to the proper temperature and remain so until one is ready to add additives and until one is done dispensing the agar.
- Polystyrene Petri dishes may deform if agar is hotter than 70°C.
- Store at room-temp for short-term use, or 4°C for long-term use and better preservation of nutrients and antibiotics, especially β-lactams and tetracyclines (amp, tet).
- For agar, dispense ≈15–20 mL into each Petri dish "plate". For agar, dispense ≈15–20 mL into each Petri dish. Work quickly to avoid agar congealing in the vessel.
After agar is set, allow excess moisture to dry for 24–48 hr before bagging in the original plate sleeves, folding the opening closed several times and clipping or taping it to hold it so for a tight seal. Store 4°C.- Pouring agar is fastest, but less accurate: stop pouring when ≈¾
- plate is covered; then let the remainder fill, slightly swirling the
- plate or stack of
- plates if needed.
- For more accuracy, use a 50 mL pipette to dispense, though slower and more prone to premature congealing and pipette clogging.
- Stacks of plates can be efficiently filled from the bottom up:
- hold open the bottommost plate while the lid supports the empty stack above it
- ; fill the open plate with the other hand holding the vessel; replace the stack+lid and continue up the stack.
- Bubbles can be eliminated by very briefly
- passing a flame over the surface.
- Plates with temp-sensitive antibiotics (amp, tet), should perhaps be refrigerated after 24 hr drying. Tet and zeo are photolabile, so dry in the dark.
Cloning Broth: LB30 enh
Enhances low- and high-copy plasmid yields (4) and is more volume-efficient. Even better with 5–10 mM Mg and (maybe) 1× M9 salts, Shyam's standard cloning broth.
LB30(regular) just has added yeast extract compared to LB; LB 30 Mg has additional Mg; LB 30 enh has additional Mg and buffering (Shyam, 6).
- 878 mL/L Milli-Q or deionized water
- Add half volume first. Rinse bottle sides with remaining half at end.
- Using scale for water saves you from measuring volume.
- 25 g/L LB powder
or from separate components:- 10 g/L tryptone
- 5 g/L yeast extract
- 10 g/L NaCl
- 25 g/L yeast extract (additionally, for final 30 g/L)
- This can probably be increased further. Gibco might be better than Bacto. See Notes.
- Autoclave 15 min for ≤1 L volumes.
After cool enough to handle, add: - 10 mM MgCl2 (autoclaved, preferred). Or MgSO4. 20 mL/L 2 M stock.
Swirl to mix; then add: - 1× M9 Salts or TB Salts or other buffer (100 mL/L 10× stock). Swirl to mix.
- I have been using old 10× M9 salts from previous lab members. Has not been tested if buffering improves "neutral" plasmid yields or just more toxic plasmid yields.
- NaCl can be omitted with use of M9 salts, if making LB from scratch.
- opt. Carbohydrate: when needed, 4–10 g/L glucose or glycerol can be added for improving growth of strains with toxic constructs. Buffering and Mg supplementation should be used with simple carbon sources (5, Notes) . May not improve plasmid yield (4, Notes) .
Cloning broth: YEMS
Yeast Extract, Magnesium, Salts. Since tryptone in LB seems to be pointless (see Notes on C/N source), this broth ought to outperform LB30 formulations above for plasmid production. Still in trial.
- 40 g/L yeast extract
- Gibco might be better than Bacto. See Notes.
- Might be able to increase even more.
- 10 mM MgCl2 (autoclaved, preferred). Or MgSO4. 5 mL/L 2 M stock.
- Salts: NaCl and/or a buffer
- 10 5 g/L NaCl, and/or
- 1× M9 Salts or TB Salts or 60–100 mM K/Na phosphate buffer pH 76.8.
If using a buffer, NaCl should perhaps be omitted , since the buffer has plenty of salt, especially M9 salts. Slight acidity helps ampicillin stability.
- opt. Carbohydrate: when needed, 4–10 g/L glucose or glycerol can be added for improving growth of strains with toxic constructs. Buffering and Mg supplementation should be used with simple carbon sources (5, Notes) . May not improve plasmid yield (4, Notes) .
Notes
Autoclaving
It has been classically suggested to separately autoclave phosphate buffers and agar, and recently it was found that phosphate and agar react to form H2O2 (9) , but E. coli are less, if at all, significantly impacted due to catalase expression. Commercial mixes do not separate the two, yet instruct autoclaving. However, phosphate also induces precipitation of trace metals at high temperatures (8) , another reason to add phosphate buffer separately after components are cool. There is also reason for separately autoclaving carbohydrate sources separately, as they partake in Maillard reactions with tryptone (8) , though glycerol probably less so than sugars do due to lack of carbonyls. Maillard reactions sequester nutrients and form products that decompose into growth-inhibitory compounds (8) .
Autoclaving ≤1 L volumes for 15 min as per CSH protocols has been sufficient for sterility. Minimizing autoclaving time reduces Maillard browning reactions that decompose nutrients and darken the medium. 1–1.5 L volumes benefit from 20 min autoclaving; agar remains cloudy with 15 min autoclaving.
Magnesium and Buffering
Magnesium is deficient in LB (2,5). It has been found that in peptide broths, supplemental Mg2+ is necessary for optimal growth, especially when a carbohydrate carbon source such as glucose is provided (5) . 1 mM MgCl2 supplementation improves E. coli, V. fischeri, and B. subtilis growth by prolonging exponential phase (5) . Contaminant Mg²⁺ in phosphate salts or other peptide broth components is evidently insufficient. In the modified Table 2 below from (5), note how supplemental Mg and buffering yield 68% higher E. coli cell density. Also note how glucose can hardly increase yield without supplemental Mg, which when added allows successful glucose utilization and drives the pH too low and arrests growth, except when additionally buffered (in LB7), enabling full glucose utilization and a whopping 11.6 final OD, 2.8-fold more than plain LB.
1 mM MgCl2 produces a slight 8% more cell yield than 1 mM MgSO4 (5: fig S1) . Glucose was used at 4 g/L, and buffering by 100 mM KHₙPO₄ pH 7. Mg supplementation was found to reduce protein acetylation by means of rerouting carbon from fermentation to biomass production (5: fig 6) .
Another study found that ≥95% of ribosome biogenesis and purine metabolism genes are downregulated by low Mg²⁺ conditions (e.g. LB), and ≥95% of flagellar genes are downregulated by sufficient/high-magnesium conditions (thus less biofilmy) (10: fig7) . Studies find "ATP synthesis is tightly coordinated with Mg²⁺ availability… cells will scavenge Mg²⁺ from ribosomes at the expense of protein synthesis before allowing intracellular Mg²⁺ to fall to levels that are insufficient to support ATP chelation. (14) "
Conclusion: 1–10 mM MgCl2 or MgSO4 can be supplemented to any peptide broth for higher cell yield and ATP and protein synthesis with less cell stress and biofilm.
Buffering Salts
M9 salts: See M9 Medium
TB (Terrific Broth) salts, 10×: 23.1 g/L KH₂PO₄ (monobasic), 125.4 g/L K₂HPO₄ (dibasic).
Notes
Carbon and Nitrogen Sources
Yeast extract was found in a study to be the main determinant of plasmid yield, with plasmid yield of LB matching that of terrific broth (TB) when yeast extract concentration was matched (4) . Neither the buffering of TB nor the better glycerol carbon source were found to improve plasmid yield. 30 g/L yeast extract was the maximum tested in this study, yielding the "LB30" above.
PDMR, a cheaper medium than LB30 enh likewise designed to maximize plasmid yield, has yet to be tested in our lab (7) , but since Terrific Broth (TB) only underperforms to PDMR ~25% in volumetric plasmid yield (7) and LB30 outperforms TB (4) , LB30 must perform even more closely to PDMR's plasmid production efficiency. TB was found to give fourfold the pUC19 plasmid volumetric yield relative to LB but at 65% the cost, also better in both respects relative to SOC (4) . PDM(R) was, however, ≈twice as cost-effectiveness as TB for a quantity of plasmid.
4–10 g/L glucose or glycerol does seem to be useful in alleviating growth inhibition of toxic DNA constructs while cloning them. It would be interesting to independently verify that adding such increases plasmid yield in general for nontoxic constructs, and does not only increase cell yield. In both experiments and cloning, magnesium and buffering ought to be used with glucose, as discussed in the Magnesium section, and the same may be extrapolated for glycerol or other carbohydrates out of caution.
E. coli oligopeptide permeases can only uptake peptides ≤650 Da (2). Yeast extracts have much more of their peptides+amino acids in this E. coli-usable range than tryptone and peptone (11, fig 2 below). This might explain why Shyam found faster and slightly higher growth in a version of LB that replaces the tryptone with additional yeast extract (fig 1 below). Superior to Bacto yeast extract ought to be Gibco yeast extract, which the manufacturer data shows is 13% higher in low-weight peptides in the <600 Da range that E. coli can eat, compared to Bacto tryptone (11). And the free amino acid data in the manual shows the Gibco YE has equal or significantly more of each free amino acid compared to Bacto YE. Also, Gibco YE has a fifth the sodium. Casamino acids are nearly completely hydrolyzed bovine casein, and though it has the most peptides in the E. coli-usable range, it has the disadvantage of being absent in tryptophan, asparagine, and glutamine, destroyed during acid hydrolysis.
Growth of E. coli DH10B in three media, all supplemented with 5 mM MgCl2 (and chl25 to maintain a plasmid):
- "LB"-Miller (5 g/L Bacto yeast extract, 10 g/L Bacto tryptone, 10 g/L NaCl)
- "tryptone" (15 g/L tryptone, 10 g/L NaCl)
- "yeast extract" (15 g/L yeast extract, 10 g/L NaCl).
From the ThermoFisher (Gibco/Bacto) Technical guide to peptones, supplements, and feeds (11)
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