LB Agar Media

Equipment & Consumables:

• PPE: Gloves, Lab Coat and Goggles

• Sterile Workspace

• Weighing Scale (accurate to to 0.01g)

  • Weigh boats (or folded paper)

• Autoclave

  • or other method for sterilisation (microwave or pressure cooker)

• Optional: Heated/Stirring Platform

• Optional: pH Meter (recently calibrated)

• Graduated Beaker

• Measuring Cylinder

• 4°C Refrigerator

• 1L ddH20 (MilliQ or RO) (400mL is optimal if autoclaving a 1L bottle)

• 10 g Tryptone (4 g per 400 ml)

• 5 g Yeast Extract (2 g per 400 ml)

• 10 g NaCl = LB-Miller (4 g per 400 ml)

  • 5 g = LB-Lennox (2 g per 400 ml)

  • 0.5 g = LB-Luria (0.2 g per 400 ml)

• 17 g Agar (6.8 g per 400 ml)

Protocol:

Recipe for 1 Litre LB-Miller Agar Media

Note: If using an Autoclave, you want lots of head room. Multiply all values by (x0.4) to fill a 1L bottle to the 400 mL mark which is the optimal amount.

1. Measure out 10g of Tryptone and add it to your bottle.

   • Note: Peptone and Tryptone are interchangeable but will have minor influences on your experiment. Feel free to use one in place of the other, but don't go swapping mid experiment.

2. Measure out 5g of Yeast Extract and add it to your bottle.

3. Measure out 10g of NaCl and add it to your bottle. (LB-Miller)

   • Note: If you plan to use everyday table salt, double check to ensure it doesn't also contain anti-caking agent. This will inhibit bacterial growth.

   • Alternative LB Recipes: 5g NaCl (LB-Lennox), 0.5g NaCl (LB-Luria)

4. Measure out 17g of Agar and add it to your bottle.

   • Note: Agar settles out at RT, rather than dissolves. Distribute your total volume between any smaller bottles if you need to do so for autoclaving before adding agar to each.

5. Add MilliQ or RO water to make volume up to 1L.

   • Note: It is possible to make LB media with tap water, but keep in mind that the mineral content will start to interfere with your experiments soon. If you don't have access to a water purification system - try hunt down a cheap purification system before it becomes a problem.

6. Tighten the lid of your bottle, then loosen by half a turn.

7. Sterilise 20 minutes/liquid cycle (121˚C) in an autoclave. Or use one of our cheaper sterilisation protocols.

8. If pouring plates today, let the bottle cool until it is ~60˚C or “warm to the touch, but no sign of solidifying” then proceed to antibiotic addition or the plate pouring protocol.

   • Note: The easiest way to do this is with a shaking water bath at 60˚C

9. If you don’t have time to hang around and pour your plates, you can let the agar-media harden, tighten the lid on the bottle and then store it in a cool, dry place.

   • Note: It is quite handy to keep a few bottles of pre-autoclaved agar-media around in case you run out of a specific type of antibiotic plate. Solid agar media in a well sealed bottle also lasts longer than some poured plates in the fridge (looking at you Ampicillin plates).

10. Loosen the lid to allow for air transfer and then reliquify the solid media in the microwave (2 x 5 minutes, Medium setting) or in a 60˚C water bath, then add your antibiotic (optional) and pour your plates.