Growth of mixed E. coli colonies
Wolfram Moebius
Abstract
Growth of mixed E. coli colonies on agar plates and between an agar plate and an agar pad
Steps
Make plates
Autoclave Lennox LB (10 g/l tryptone / casein digest peptone, 5 g/l NaCl, 5 g/l yeast extract) with 1.5 % agar (w/v).
Optional: Add antibiotics at desired concentration after medium has cooled down sufficiently.
Pipette 10mL of LB and agar in Petri dishes with diameter of 6 cm .
Leave plates plates to dry at 4Room temperature. Optionally enclose to avoid nonuniform drying.
Store and prepare plates
If not used the day after pouring plates, store at 4°C. Enclose in plastic container or bag to avoid further drying of plates.
Before usage of plates that have been refrigerated, warm up plates at 37°C.
Prepare bacterial cultures
Grow overnight culture (colony picked or directly from glycerol frozen stocks) in Lennox LB.
Inoculate colonies and incubate
Mix 900µL Lennox LB and 50µL of each of the two overnight cultures in an Eppendorf tube.
Vortex.
Inoculate 1µL in centre of plate and let dry.
Optional: Cover colonies with agar pad. Cut about 18 mm x 18 mm agar pad inside plate. Use spatula to lift pad. Place vertically next to colony and let fall upside down onto colony. Use spatula to remove bubbles by pressing on top.
Place plates into container, together with sufficiently wet paper towels. Incubate for 7-8 days in a dark environment.
