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Agar and Plate Preparation

 

  • 1000 mL Minimal Media (1 mm KCl, 1mm CaCl2, 5mm MES (methyl-ethyl-sulfate)
  • 2000 mL Erlenmeyer flask
  • 1 graduated cylinder
  • Difco agar, granulated
  • 1 Weighing boat
  • Autoclave and Tinfoil
  • ≈130 petri dishes
  • Hot plate
  • 60 mL syringe
  • stop-cock attachment
  • 70 % EtOH
  • Large storage containers

 

2) Agar and Plate Preparation

 2.1)  Measure out 1000 mL of Minimal Media (1 mM KCl, 1mM CaCl2, 5mM MES (methyl-ethyl-sulfate), pH 5.7 with BTP (bis-tris propane), 1% agar) in a graduated cylinder then pour into a 2000 ml Erlenmeyer flask. Measure 300 more ml of media in the graduated cylinder.
 2.2) Weigh out 13 g of the Difco Agar, Granulated and add to the Erlenmeyer flask. DO NOT weigh extra.
 2.3) Use the remaining 300 ml of the Minimal Media to get the last of the granulated agar from the weigh boat. Swirl the solution in the Erlenmeyer flask so that it dissolves as much as possible.
 2.4) Put tin foil on the top of flask and place it in the Autoclave.
 2.5) Set the Autoclave to the liquid #2 setting with the temperature at 121°C and time at 12-15 minutes.
 2.6) While the agar solution is in the autoclave, set up approximately 130 Petri dishes to pour the agar into when it is done in the autoclave.
 2.7) After the solution is done autoclaving, place it on a heating plate set at 89°C to prevent solidification.
 2.8) Rinse a 60 mL syringe and stop-cock valve with a 70% ethanol solution for sterilization prior to plate preparation.
 2.9) Pour the agar solution into the sterilized syringe (total 60 mL) with stop-cock attachment. Leave the syringe plunger out.
2.10)  Pour 10 mL of agar into each petri dish. The 60 mL syringe will only allow the pouring of 6 dishes.
2.11) After 6 plates are poured, swirl the plate filled agar so that it completely covers the bottom, and place the lid on top. Hold the plate a few inches off the preparation surface to prevent scratching of the dish. 
2.12)  Repeat steps 2.9-2.11 until the agar runs out.
2.13) After all of the plates are poured, swirled, and covered let them sit for a couple hours so the agar can solidify.
2.14) Place petri dishes in containers to prevent the agar from drying out.