Materials

Consumables for 1L of media:

  • 900 mL Distilled Water
  • 20 g Peptone
  • 10 g Yeast Extract
  • 20 g Agar
  • 100 mL (bottle) 20% glucose
  • Plastic Weigh Boat
  • Aluminum Foil
  • Autoclave Tape
  • 0.2μm filter
  • 3 mL syringe

Hardware:

  • 2L Flask
  • Stir bar
  • Hot plate
  • Stir bar magnet stick
  • Autoclave tub
  • Autoclave

Media Creation

  1.  Measure out calculated volume of distilled water in a 1L graduated cylinder
  2.  Pour into flask on hot plate and add stir bar
    • Use 2L flask for 1L of media
  3.  Start stir bar spinning and gradually increase spinning
  4.  Using a plastic weight boat, measure the appropriate amount of dry reagents and pour into flask
  5.  Allow for the mixing of all the reagents. This is complete when there are no more clumps of powder. Turn up the stir bar spinning if necessary.
  6.  Retrieve the stir bar with the magnetic stick, rinse, and place in autoclave-able box with piece of autoclave tape
  7.  Place a square of aluminum foil over the mouth of the flask
  8.  Add a small strip of autoclave tape to the top of the flask and stir bar box
  9.  Place the stir bar box and the flask in the autoclave tub
  10.  Place the tub in the autoclave and set to ‘Liquid 30’ setting

Adding Antibiotics

Calculate the amount of stock needed per liter to produce your desired concentration by using this conversion calculator.

You must know:

  • Concentration of stock (in mg/mL)
  • Desired final concentration (in ug/mL)

You will be finding the volume of stock needed per liter to create the desired final concentration.

Final Additions

  1.  After autoclave cycle put on stir plate and add autoclaved stir bar
  2.  Begin spinning stir bar, slowly pour glucose down the side of flask into media
  3. Pipet calculated amount of antibiotic stock solution (if adding antibiotic) into media and allow to mix until combined
  4. Pour prepared media into sterile 1L bottle and place in fridge