May 27, 2022
Preparing LB Broth or Agar +/- Antibiotic
This protocol is a draft, published without a DOI.
- 1Mboalab, Beneficial Bio
Protocol Citation: Stephane Fadanka, Mujar Minette Shalo, Nadine Mowoh 2022. Preparing LB Broth or Agar +/- Antibiotic. protocols.io https://protocols.io/view/preparing-lb-broth-or-agar-antibiotic-b85qry5w
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: May 10, 2022
Last Modified: May 27, 2022
Protocol Integer ID: 62352
Keywords: LB Broth, LB Agar
Abstract
In order for bacteria to be successfully cultured, they must be grown in the appropriate media. LB, also known as Lysogeny broth (also known as Luria broth, Lennox broth, or Luria-Bertani medium.), is a nutrient rich broth that is a standard for culturing Escherichia coli, as it allows for quick growth and high yields. Furthermore, addition of agar to LB broth creates a gel for bacteria to grow upon, and is therefore used for plating bacterial cultures on petri dishes for single colony isolation.
This procedure is to be used for the preparation of LB media and LB plates
+/- antibiotic.
The formulations generally differ in the amount of sodium chloride, thus providing selection of the appropriate osmotic conditions for the particular bacterial strain and desired culture conditions. The low salt formulations, Lennox and Luria, are ideal for cultures requiring salt-sensitive antibiotics.
LB-Miller (10 g/L NaCl)
LB-Lennox (5 g/L NaCl)
LB-Luria (0.5 g/L NaCl)
This SOP includes instructions to make either 1litre or 120ml LB broth.
Materials
Chemicals
- Yeast Extract
- Tryptone
- NaCl
- Agar
- Antibiotic stock solution (as required)
Equipment
- Beaker
- Measuring cylinder
- Electronic balance
- Weighing boat
- Spatula
- Aluminum foil
- Distilled water
- Clean Erlenmeyer Flasks (50ml and 250ml)
- Sterile Petri dishes
Safety warnings
The process is generally safe but safety lab procedure must be followed and avoid dust production and inhalation form the media.
Before start
Ensure you check that all the materials and reagents required for this process are available and assembled.
Weighing components for LB Broth
Weighing components for LB Broth
Weight all powders Yeast Extract CAS 8013-01-2, NaCl CAS 7647-14-5 and Tryptone CAS 91079-40-2 as indicated in the table below using weighing boats and place powders into an appropriate size beaker, add the corresponding amount of dH2O and mix using a magnetic stirrer.
| LB media composition Reagent | Amount for 1L (g) | Amount for 120ml (g) | |
| Yeast Extract | 5 | 0.6 | |
| NaCl | 5 | 0.6 | |
| Tryptone | 10 | 1.2 |
Dispense the resulting 120ml mixture into the following flasks;
2x 50ml LB broth in 250ml conical flasks
1x 5 ml and 1 x 10 ml in 50 ml conical flask
Seal flasks with aluminum foil and autoclave to make the culture media sterile
Remove flasks from autoclave, label with production date and store at room temperature or +4°C until required.
Note
Add appropriate antibiotic as required prior to use
Weighing components for LB Agar Plates
Weighing components for LB Agar Plates
Weight all powders in a beaker as indicated in the table below, add the corresponding amount of dH2O and mix using a magnetic stirrer (all will dissolve apart from agar).
| LB media composition Reagent | Amount for 1L (g) | Amount for 120ml (g) | |
| Yeast Extract | 5 | 0.6 | |
| NaCl | 5 | 0.6 | |
| Tryptone | 10 | 1.2 | |
| Agar | 15 | 2.4 |
Complete steps 2 and 3 above.
Remove the flasks from the autoclave and allow the media to cool to about 55°C (until they can be held by hand)
Add appropriate antibiotic, if required, prior to use
Adding Antibiotic
Adding Antibiotic
The case of Kanamycin 50µg/ml, Kanamycin Sulphate powder CAS 25389-94-0
50µg/ml final concentration Kanamycin (add 0.5µl per ml LB agar from a 100mg/ml stock solution prepared using this protocol).
Aliquoting LB Agar into Petri dishes
Aliquoting LB Agar into Petri dishes
Promptly and smoothly pour LB Agar medium +/- antibiotic (approx. 15-20ml) into the base of Petri dishes, cover with Petri lid and allow to cool and completely solidify.
Label Petri dishes with manufacture date and antibiotic and store at +4°C until they are used if not used immediately.
Note
To avoid contamination, plates should be discarded if not used after 1month.