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Microbiology - Plating in Top Agar

Microbiology - Plating in Top Agar


Jeff Lawrence

1. Warm plates to room temperature before use. Cold plates causes the top agar to solidify irregularly. DO not warm plates to 37° as the top agar will take forever to solidify.

2. Prepare top agar as the appropriate liquid medium with 0.7% agar. Keeping 100 mL bottles is convenient. For phages, use λ top agar, which is less rich and yields bigger plaques.

3. Melt top agar in the microwave completely. Allow the agar to boil after liquification; incompletely melted agar looks liquid, but is grainy.

4. Allow agar to cool to 48°. Store in the 48° waterbath for convenience; old agar turns dark and should be discarded. Agar with drugs cannot be stored.

5. Add cells and phage to a 13 mm yellow-capped tube in the 48° temperature block.

6. Remove the cap and add 2.5 mL molten top agar. Do not replace cap.

7. Immediately vortex the mixture, flame the top, and dispense the mixture onto the appropriate plate. Swirl the plate to evenly distribute the agar.

8. Allow the plate to stand for several minutes as the agar cools.

9. After the top agar is solid, invert plate and incubate.