Protocols

LB Agar Plates

Making LB Agar Plates - 600mL/25 plates
Ingredients:
6 g Tryptone
3 g NaCl
3 g Yeast Extract
0.6 mL of 1 M NaOH (600 μL)
9.0 g Agar
Milli Q Water to 600 mL

Or

12 g of premixed LB
0.6 mL of 1 M NaOH
9.0 g agar
MilliQ water to 600 mL

Procedure: Using a large scoopula measure out the above powders into a tared weigh boat on an analytical balance. The NaOH can be taken out of the bottle with a micropipetman. The LB agar does not have to be precisely measured to a high number of significant figures but should be close to these values. After addition of the powders and base into the 1000 mL Wheaton bottle, Milli Q water can be added to the 600 mL gradation. Screw the lid on and mix the solution.

After some mixing loosen the lid and add a small piece of autoclave tape to the top of the bottle. The autoclave tape should have a small tab on it so that it can easily be removed later. The bottle should be put in a white tub and both should be put into the autoclave machine. The autoclave should run the agar solution through a liquid cycle for 20 minutes and then removed and allowed to cool to 50°C. At around 50°C we want to add an appropriate amount of antibiotic to the agar. At 50°C the solution will still be hot to the touch but you should be able to pick it up and hold it.

After adding the antibiotic, quickly pour the agar into a sleeve of plates. The sleeve should be labeled with a stripe along the side to signal which antibiotic was used. 600 mL will be just enough for an entire sleeve so don’t use too much for any one plate. For each plate, pour the solution until it reaches all edges of the plate, and then stop and cover with the lid. The solution will take an hour or so to cool.