OVERNIGHT CULTURES

An overnight (ON) culture grows a single bacterial colony in liquid broth for about 12–16 hours, producing a dense, fresh culture for the next day’s experiment. An antibiotic can be added to keep only bacteria that carry a resistance plasmid.

WHAT YOU NEED?

• Sterile LB broth

• Sterile culture tubes

• Antibiotic stock, if needed (for example tetracycline at 20 µg/mL)

• Plate with isolated colonies

• Sterile loops, serological pipette and micropipette

• Shaking incubator at 37°C

STEPS

1. Label the tubes, including one negative control tube.

2. Add 5 mL of LB broth to each tube using a sterile serological pipette.

3. Add antibiotic to the tubes that need it, using a micropipette.

4. With a sterile loop, pick one well-isolated colony and swirl it into the broth. Use a new loop for each tube.

5. Leave the negative control tube uninoculated (or dip in an unused sterile loop) to check for contamination.

6. Place the tubes in a shaking incubator overnight (about 12–16 hours).

7. Next day, check that inoculated tubes are cloudy and the negative control is clear.

TIPS

• Always start from a single colony so the culture is pure.

• Don’t leave cultures much longer than 16 hours; the cells start to die off.

• A cloudy negative control means contamination, so discard that batch.