MEDIA PREPERATION

LB (Lysogeny Broth) is a nutrient-rich medium used to grow E. coli and many other bacteria. LB broth is the liquid version for overnight cultures; LB agar is the same recipe with agar added so it sets into a solid gel for plates.

WHAT YOU NEED?

• Tryptone (10 g per litre)

• Yeast extract (5 g per litre)

• NaCl (5 g per litre)

• Agar (15 g per litre, for plates only)

• Deionised (DI) water

• Media bottles, balance, weigh boats and a stir bar

STEPS

1. Fill around 750 mL of DI water in a graduated cylinder.

2. Weigh out the dry ingredients (10g Tryptone + 5g NaCl + 5g Yeast Extract).

3. Add them to DI water and mix until dissolved.

4. Top up to the final volume with DI water.

5. Divide into media bottles, filling each no more than 200mL full.

(For LB Agar add 3g of Agar before filling the bottles)

6. Loosen the caps and autoclave on a liquid cycle (121°C, 20 minutes).

7. Let it cool, tighten the caps, label with name and date.

TIPS

• Add antibiotics only after the media has cooled; heat breaks them down.

• Label every bottle with the medium, any antibiotic, the date and your initials.

• Solid LB agar can be re-melted later by steaming (see Steaming Media).