May 07, 2020
Growing overnight culture of OP50 as worm food
- 1Imperial College London
- Behavioural Genomics
Protocol Citation: Priota Islam 2020. Growing overnight culture of OP50 as worm food. protocols.io https://dx.doi.org/10.17504/protocols.io.bf5pjq5n
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 07, 2020
Last Modified: May 07, 2020
Protocol Integer ID: 36751
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 protocol is specifically for making a liquid bacterial culture of OP50 and the following parameters are specific for this bacterial strain:
1. Name of the bacterial strain: E.coli (OP50)
2. Growth temperature: 37C
3. Incubation time: 16-18hrs (Overnight)
4. Rpm of the shaking incubator: 200-220 rpm
Obtain LB Broth from the Media kitchen- Make sure to make at least 500ml in volume for standardisation purposes
LB Broth contents:
4gNaCl
4 g Tryptone
2 g Yeast Extract dH2O to 400 mL
Obtain sterile Erlenmeyer flask from the media kitchen
Wipe the work area with 70% ethanol and create a relatively sterile environment on the laboratory bench by using a simple gas-powered burner
Add the desired volume of liquid LB to the flask
Using a sterile inoculation loop, select a single colony from your bacteria streaked LB agar plate (One streaked plate can be used for about a month and stored at 4C during that time period)
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 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