Ingredients (needed quantity)

• LB Agar (Miller Mod) 150mL in a 250mL Bottle
• Kanamycin 100mg/mL Stock Solution
• Gentamicin 100mg/mL Stock Solution
• Rifampicin 15mg/mL Stock Solution
• Sterile Petri Dishes

Equipment

• Pipettes
• Microwave Oven
• Hot mitts or heat protection for hands
• Laminar Flow Hood or Aseptic Conditions

Step By Step

1. Partially unscrew the cap of a 250mL Pyrex bottle containing 150mL of LB Agar (Miller Mod).
2. Place the bottle in the microwave. BE SURE THE CAP IS PARTIALLY UNSCREWED AND CAN BE RATTLED UP AND DOWN BUT DOES NOT COME OFF. Microwave until molten. This may require some initial trials to see what your microwave oven’s energy and time requirements are. In my small microwave, I can melt it in 10 minutes but in a standard kitchen microwave it can melt in far less time. It’s better to run on lower power for longer to avoid boil over. Write down the ideal power and time parameters as these will be handy. Melting parameters will vary with ambient temp and weather conditions outside.
3. Carefully remove the molten agar bottle from the microwave using proper PPE and heat resistant gloves and place them in your laminar flow hood or aseptic area.
4. Allow the bottle to cool to about 55C which is roughly the hand feel of a very hot cup of coffee that you are still willing to drink. You should be able to hold the bottle for 10 seconds without discomfort when at this temperature. BE CAREFUL NOT TO BURN YOURSELF.
5. Once cool, add 150uL of Kanamycin 100mg/mL Stock Solution
6. Add 150uL of Gentamicin 100mg/mL Stock Solution
7. Add 150uL of Rifampicin 15mg/mL Stock Solution
8. Swirl the bottle for 10 seconds to mix the antibiotics.