Growing overnight culture of OP50 as worm food
Bonnie Evans
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:
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Name of the bacterial strain: E.coli (OP50)
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Growth temperature: 37C
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Incubation time: 16-18hrs (Overnight)
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Rpm of the shaking incubator: 200-220 rpm
Steps
Obtain 100mL LB Agar from the media kitchen. Heat in the microwave in 20s bursts until liquid.
Label 2 x 90mm Petri dishes with 'LB Agar' and the date. Using a stripette, dispense 35mL LB agar into each of them. Leave to set on bench.
Streak OP50 onto LB Agar and grow overnight at 37C.
The next day, check for individual colonies on OP50 plate. Parafilm the plate and store in 4C fridge for up to a month.
Obtain 1L LB Broth from the media kitchen
LB Broth contents:
4gNaCl
4 g Tryptone
2 g Yeast Extract dH2O to 400 mL
Obtain 2 x 2L sterile Erlenmeyer flask from the glassware room
In the microbial hood, add 500mL liquid LB to each flask
Using a sterile pipette tip, select a single colony from your plate. Drop pipette tip into the flask.
Re-cover the flask with the sterile aluminium foil
Incubate the bacterial cultures at the 37C 200rpm for 16-18 hours.
After incubation, check for growth, which is characterized by a cloudy haze in the media.
Measure the optical density of the overnight culture.
Optical density measurement for Bacterial solution (Worm Food)
Adjust OD600 of the culture to 1.0 using LB.
Amount of LB (mL) to add = (OD600 x 500mL) - 500mL
Aliquot culture into 50mL Falcon tubes and store in 4C fridge.
