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Protocol CitationAndreas Sagen 2023. Lysogeny Broth (LB) medium. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1o262lr2/v2Version created by Andreas Sagen
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 06, 2023
Last Modified: January 06, 2023
Protocol Integer ID: 74863
Keywords: LB, bacteria, Lennox broth, E. coli, Escherichia coli
Abstract
Lysogeny broth (LB) is a nutritionally rich medium which is primarily used for the growth of bacteria[1]. The initialism is also commonly, albeit incorrectly, taken to mean Luria broth, Lennox broth, or Luria-Bertani medium. According to its creator Giuseppe Bertani, the abbreviation LB was actually intended to stand for lysogeny broth. The formula of the LB medium was
published in 1951 in the first paper of Bertani on lysogeny[2]. There are several common formulations of LB. Although they are different, they generally share a somewhat similar composition of ingredients used to promote growth, including the following: Peptides and casein peptones, Vitamins (including B vitamins), Trace elements (e.g. nitrogen, sulfur, magnesium) and Minerals.
Sodium ions for transport and osmotic balance are provided by sodium chloride (NaCl). Tryptone is used to provide essential amino acids such as peptides and peptones to the growing bacteria, while the yeast extract is used to provide a plethora of organic compounds helpful for bacterial growth. These compounds include vitamins and certain trace elements.
Guidelines
Follow step by step, unless stated otherwise. Equipment needed should be standard to a microbiology lab.
Materials
Analytical scale, autoclave, bottle, weight vessel, LAF bench
Protocol materials
ReagentTryptoneMerck Millipore (EMD Millipore)Catalog #T9410
In 2 steps
ReagentYeast ExtractMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0875
In 2 steps
ReagentSodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9625
In 2 steps
ReagentAgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
Step 2.2
Safety warnings
When removing autoclaved components, be sure to take care as this can be very hot. If using antibiotics, use sufficient PPE to protect yourself, as some can be toxic to humans.
Before start
Prepare glassware by cleaning it, and ensure that scale is sufficiently calibrated
500 mL LB-Lennox (broth) medium
500 mL LB-Lennox (broth) medium
All compounds are measured using a high precision analytical scale from powdered compounds. Each compound is measured to within 1% of the target weight. All compounds are mixed in a Duran bottle
Fill the bottle with Amount400 mL double-distilled water
Measure Amount5000 mg Tryptone, Amount2500 mg Yeast extract and Amount2500 mg Sodium chloride

Powdered compounds:
ReagentTryptoneEmd MilliporeCatalog #T9410
ReagentYeast ExtractEmd MilliporeCatalog #Y0875
ReagentSodium chlorideEmd MilliporeCatalog #S9625
Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for Duration00:05:00
5m
Adjust pH while mixing to Ph6.7 using concentrated sodium hydroxide
Add distilled water to a total of Amount500 mL
Autoclave liquid at Temperature121 °C for Duration00:15:00
Note
Cool to 50°C and supplement with antibiotics as appropriate

15m
500 mL LB-Lennox (agar) medium
500 mL LB-Lennox (agar) medium
20m
20m
All compounds are measured using a high precision analytical scale from powdered compounds. Each compound is measured to within 1% of the target weight. All compounds are mixed in a Duran bottle
Fill the bottle with Amount400 mL double-distilled water
Measure Amount5000 mg Tryptone, Amount2500 mg Yeast extract, Amount2500 mg Sodium chloride and Amount7500 mg agar

Powdered compounds:
ReagentTryptoneEmd MilliporeCatalog #T9410
ReagentYeast ExtractEmd MilliporeCatalog #Y0875
ReagentSodium chlorideEmd MilliporeCatalog #S9625
ReagentAgarEmd MilliporeCatalog #A1296
Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for Duration00:05:00
5m
Adjust pH while mixing to Ph6.7 using concentrated sodium hydroxide
Add distilled water to a total of 500 mL
Autoclave liquid at Temperature121 °C for Duration00:15:00
Note
Cool to 50°C and supplement with antibiotics as appropriate

Agar can be stored, then reheated to 50°C to be poured

15m