Oct 25, 2019
Inoculating a Liquid Bacterial Culture
- 1Imperial College London
- Behavioural Genomics
Protocol Citation: Saul Moore, Ida Barlow 2019. Inoculating a Liquid Bacterial Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.8rxhv7n
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: October 25, 2019
Last Modified: October 25, 2019
Protocol Integer ID: 29207
Abstract
Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. LB agar plates are frequently used to isolate individual (clonal) colonies of bacteria carrying a specific plasmid. However, a liquid culture is capable of supporting a higher density of bacteria and is used to grow up sufficient numbers of bacteria necessary to isolate enough plasmid DNA for experimental use. The following protocol is for inoculating an overnight culture of liquid LB with bacteria.
This is a general protocol for making a liquid bacterial culture and the following parameters need to be mentioned for a specific type of bacterial strain:
1. Name of the bacterial strain
2. Growth temperature
3. Incubation time
4. Antibiotic resistance (if any)
5. Rpm of the shaking incubator
Obtain LB Broth from the Media kitchen
LB Broth contents:
- 4 g NaCl
- 4 g Tryptone
- 2 g Yeast Extract
- dH2O to 400 mL
Add liquid LB to a tube or flask and add the appropriate antibiotic (if required) to the correct concentration (see table below).
Antibiotic Concentrations
| Commonly Used Antibiotics | Recommended Concentration | |
| Ampicillin | 100 µg/mL | |
| Bleocin | 5 µg/mL | |
| Carbenicillin | 100 µg/mL | |
| Chloramphenicol | 25 µg/mL | |
| Coumermycin | 25 µg/mL | |
| Gentamycin | 10 µg/mL | |
| Kanamycin | 50 µg/mL | |
| Spectinomycin | 50 µg/mL | |
| Tetracycline | 10 µg/mL |
Antibiotic concentrations
Note: If you intend to do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might want to use as much as a litre of LB in a 2 L Erlenmeyer flask.
Using a sterile inoculation loop, select a single colony from your bacteria streaked LB agar plate.
Dip the inoculation loop into the liquid LB and swirl.
Discard the inoculation loop.
Loosely cover the culture with sterile aluminium foil or a cap that is not air tight as bacteria needs air.
Incubate the bacterial culture at the required growth temperature overnight (i.e. 12-18 hrs in general) in a shaking incubator.
After incubation, check for growth, which is characterized by a cloudy haze in the media.
Measure the optical density of the bacterial culture at 600nm wavelength using a spectrophotometer.
Record the OD600 three times and calculate average, use LB Broth as Blank.