Pouring Plates Protocol:

1. Microwave LB or TB media until completely liquid (be careful of rapid phase changes called ‘superheating’). For 250mL, 3 - 4 minutes on high power or 12 - 14 minutes on 30% (low)
2. Prep plating area: sterilize surface with ethanol, turn bunsen burner on
3. If antibiotic: (stocks are 1000x) place 1:1000 ratio.
example: if media is 250 mL, place 250 uL in broth
4. Lay out ready to go plates
5. Pour in ~ 20 - 30 ml of agar per plate
6. Partially close/lay the lids over the cooling plates (everything near the burner)

Plating Protocol 2:
1. Place the Petri plates to be poured right side up in front of you (if many plates are to be poured, you may want to set them up in stacks of three).
2. Obtain a bottle of agar. Microwave agar with no lid. For less volumes approx. 1min per 100ml (full power). For 250ml 15mins at 30% power - this avoids agar boiling over.

  • Let cool until you can hold bottle in hand.
  • Add antibiotics 1ul per 1ml.

3. Pour 10-15 mls of molten agar into each plate (Fig A-3b). Stop just before the pool of agar touches the edges of the plate. With practice you will learn how to judge the proper amount by eye. Be careful to prevent the drip on the lip from running down onto the contaminated outer glass.
4. Immediately after pouring, cover the plates and use a firm circular motion to uniformly distribute the agar over the plate. Be quick or the agar will solidify before mixing.
5. Carefully slide the plates to an undisturbed area of the bench to solidify. Once solidified, invert the plates, place in sleeve and label well with tape.

Figure A-3: (a) Removal of a bottle cap aseptically. Note how the rest of the hand is free to manipulate a pipette or plate lid. (b) Pouring agar into a plate. Note how the lid shields the agar from airborne contamination.