Microbial Growth Protocols

Culture of E. coli

Materials required:

Suspension culture of E. coli

1. Prepare LB medium by weighing appropriate powder medium and adding to water in a sterile flask
2. Autoclave the broth and cool to room temperature
   (Alternatively ready-to-use LB medium may be used)
   
3. In a laminar flow chamber, transfer approximately 1 mL of overnight E. coli culture to the flask
4. Seal the mouth of the flask with sterile cotton (non-absorbant) plugs; ensure the flask is not tightly sealed
5. Incubate overnight at 37° C with continuous shaking

Plating E. coli

1. Prepare LB agar by weighing appropriate powder medium, agar and water to a sterile flask
2. Autoclave the medium and cool just enough to be able to handle the flask
3. In a laminar flow chamber, pour approximately 25-30 mL of the LB agar into sterile plates
4. Allow the plates to set in the laminar flow chamber with lids slightly opened
   (The plates may also be stored inverted at 4° C for future use)
   (Alternatively, ready-to-use LB agar plates may be used)
   • Lightly scratch the surface of frozen E. coli glycerol stock with a sterile inoculating loop
   • Pick up E. coli colony from a plate with culture with a sterile inoculating loop
   • Add 10-100 µL of E. coli suspension culture and add one the LB agar plate
     • Streak the loop across the LB agar plate
     • Spread the culture all over the plate using a sterile glass spreader
5. Invert and incubate the plates overnight at 37° C