Mar 11, 2026
Lysogeny broth (LB) Agar Plate Preparation
This protocol is a draft, published without a DOI.
- Tamra Lahom1
- 1Carleton College
Protocol Citation: Tamra Lahom 2026. Lysogeny broth (LB) Agar Plate Preparation. protocols.io https://protocols.io/view/lysogeny-broth-lb-agar-plate-preparation-jvz9cn797
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: March 11, 2026
Last Modified: March 11, 2026
Protocol Integer ID: 313121
Keywords: agar plate preparation luria broth, lysogeny broth, gel that bacteria, nutrition from the lb, culture bacteria in the lab, antibiotic, culture bacteria, antibiotic of interest, addition of an antibiotic, agar plate, bacteria, bacteria with resistance, own lb, antibiotic resistance gene, lb results in the formation, lab, addition of agar, luria, lb result, lb, plasmid, nutrition
Abstract
Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. The addition of agar to LB results in the formation of a gel that bacteria can grow on, as they are unable to digest the agar but can gather nutrition from the LB within. The addition of an antibiotic to this gel allows for the selection of only those bacteria with resistance to that antibiotic - usually conferred by a plasmid carrying the antibiotic resistance gene. The following protocol will allow you to make your own LB/agar plates with your antibiotic of interest.
Troubleshooting
Collect Glassware
Collect a1 L bottle with top.
Ensure the bottle is clean by rinsing with DI H2O
Combine Media
To prepare 0.5 L, dissolve5 g tryptone, 2.5 g yeast extract, 5 g NaCl , and7.5 g of agar in 0.250 L of deionized water and fill to 0.5 L
Note
Have a little water in the bottle before you add the dry powder.
Autoclave
Place autoclave tape on the lid of the bottle and autoclave at 121°C for 20-30 minutes
Note
Make sure the lid is loosened to avoid pressure build up.
Pouring Plates
Place empty sterile plates around a flame. Allow the LB media to cool to < 50 C
Allow the LB media to cool to <50 C then add the appropriate antibiotic (see next section)
Note
The media is sterile at this point. DO NOT insert a thermometer. When bubbles remain when it swirls it is around the right temperature.
Pour the media into the plates and allow it to solidify before closing the lid completely
Store upside down.
Label the plates with the date it was poured and the antibiotic(s) it contains
Antibiotic Concentrations
| A | B | C | |
| Antibiotic | |||
| Ampicillin | 100 mg/mL | 100 µg/mL | |
| Bleocin | 5 mg/mL | 5 µg/mL | |
| Carbenicillin* | 100 mg/mL | 100 µg/mL | |
| Chloramphenicol | 25 mg/mL (dissolve in EtOH) | 25 µg/mL | |
| Coumermycin | 25 mg/mL (dissolve in DMSO) | 25 µg/mL | |
| Gentamycin | 10 mg/mL | 10 µg/mL | |
| Kanamycin | 50 mg/mL | 50 µg/mL | |
| Spectinomycin | 50 mg/mL | 50 µg/mL | |
| Tetracycline | 10 mg/mL | 10 µg/mL | |
Unless otherwise indicated, the antibiotic powder can be dissolved in dH2O.
*Carbenicillin can be used in place of ampicillin. Carbenicillin is more stable, so it is potentially more effective at selecting only bacteria containing the plasmids of interest (for example, fewer satellite colonies will grow). However, it is also more expensive.
Storage
Store at 4 C until use.
Note
If the plate noticeably changes color, appears dehydrated, or has growth immediately dispose in the biohazard waste.