Plating and Colony Counting

  1. Remove liquid culture from incubator.
  2. Vortex culture for a few seconds.

Making Serial Dilutions

  1. Label lid of microtitre plate with culture name and dilutions (100, 10-1, 10-2, etc.).
  2. Pipette 90 μL of 1X PBS into each well on microtitre plate, except well(s) for undiluted culture (i.e. 100 dilution).
  3. Pipette 100 μL of culture into each 100
  4. Extract 10 μL of culture from 100 well and pipette into 10-1 well (already containing 90μL of PBS); aspirate 3-5 times to and verify that all contents of the pipette tip are expelled. Discard pipette tip.
  5. With pipette volume set at 75 μL, aspirate contents in 10-1 well 3-5 times to ensure thorough mixing. Discard pipette tip.
  6. Repeat steps 6 and 7, extracting 10 μL of diluted culture from 10-1 well and pipetting into 10-2
  7. Continue 10-fold dilution until desired dilution has been reached.

Spot Plating

  1. Label base of LB plate with culture name and dilutions (see sample in Figure 1).
  2. Extract 10 μL of culture with highest dilution (i.e. lowest cell concentration) from microtitre plate and pipette onto LB plate, in area labeled with corresponding dilution. Extract another 10 μL of culture of same dilution and pipette onto LB plate, next to the first 10 μL. Make sure that the two spots do not touch each other. (Depending on the experiment at hand, you may wish to do more than 2 replicate spots.)
  3. Repeat step 2, transferring culture from microtitre plate to LB plate, in order of decreasing culture dilution (i.e. increasing cell concentration). By proceeding from highest dilution to lowest dilution, the same pipette tip may be used throughout the plating process.
  4. After plating is complete, leave plates on bench top to air dry.
  5. Invert plates and incubate overnight.

Spread plates and spot plates 1.mp4

Spread plates and spot plates 2.mp4

Spread plates and spot plates 3.mp4

Spread plates and spot plates 4.mp4

Spread plates and spot plates 5.mp4

Spread plates and spot plates 6.mp4

Spread plates and spot plates 7.mp4

Spread plates and spot plates 8.mp4

Spread plates and spot plates 9.mp4

Colony Counting

Count number of colonies resulting from each spot. Plates overgrown with colonies can be reported as too numerous to count (TNTC).



Counting Colonies.mp4

Plating Info 1.mp4

Plating Info 2.mp4

Plating Info 3.mp4