Plating and Colony Counting

Making Serial Dilutions

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

Label base of LB plate with culture name and dilutions (see sample in Figure 1).
Extract 10 mL 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 mL of culture of same dilution and pipette onto LB plate, next to the first 10 m 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.)
Repeat step 11, 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.
After plating is complete, leave plates on bench top to air dry.
Invert plates and incubate overnight.