Protocols/Pouring plates

Abstract

You need plates. Time to pour some.

Materials

For 1 L of Media:

• 10 g of Agar (for 1% Agar plates (w/v))
• media components specific to media of interest
• 1 L ddH2O
• Sterile antibiotic stock solution (if required)
• Sterile Plates

Equipment

• Autoclave

Procedure

Mixing and Sterilizing Media

1. Add dry materials and H2O to a flask sufficiently large to minimize boil-over. (ex.2 L flask for 1 L of media.)
2. (optional, really unnecessary) add a magnetic stir bar and stir the media.
3. Cover the top of the flask LOOSELY with aluminum foil.
4. Autoclave the media on the Fluid Cycle, 30 min, 255 deg C. (Keep in mind autoclave reduces pressure slowly. It will take more time than 30 min to execute the cycle.)
5. Remove media to 55 deg. H2O bath or room temp to cool.
6. Once media has cooled to ~55 deg. C, add antibiotics, if required.

Pouring Plates

1. Obtain 2 sleeves of empty plates. 1 L of media will make about 40 plates.
2. Using sterile technique, pour the media into the plates.
   • Cover the base of the plate, and then just a bit more after that.
3. Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e., more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. (See a demo on this). Another way to remove the fine bubbles that may be in your flask before puring is to mist the inside of the flask with a 75% ethanol spray bottle.
4. Leave plates to dry and cool for a while (overnight even).
   • It is a good idea to label the stack of plates to indicate antibiotic.
5. Store the plates in their original bags - upside down, so that the gel is hanging downwards (this keeps condensation off the gel).
6. Label the bags following lab convention
7. Store the labeled bags of plates in the cold room.

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.

Notes

Labeling plates can be confusing. Hear are some hints to suggest some rules and order.

• Use a consistent color to indicate antibiotic
  • Red=Kan
  • Black=Amp
• Use a sharpie to mark the sides
  • 1 line = 10 μg/mL.
  • 4 lines means 40 μg/mL. Clever ehh?
• Also write name of antibiotic, and concentration, on bag

Specific Protocols

• Endy:Pouring_plates
• IGEM:Brown/2007/Lab_Protocols/Pouring_Plates_2
• Lissa1:Pouring_Plates
• Endy:Pouring_LB_plates_from_prepped_media
• NanoBio:_LB_plates

Discussion

You can discuss this protocol.