McClean: Frogging a Serial Dilution
Frogging a serial dilution onto solid media is an effective way to distinguish and compare viability under certain growth conditions, etc. The serial dilution helps to eliminate the possibility that a relatively high cell concentration could be misinterpreted as healthy growth, for example.
- 96-well plate (flat bottom wells)
- Sterile petri dish about half-full of 95% ethanol (This NEEDS to be GLASS!!!)
- Sterile petri dish about half-full of sterile water
- Multi-channel pipettor preferably with a range of 20 to 200 μL
- Take your cells directly from culture (can be overnight or subculture) and load 200 μL of each sample into a well in the first column (left-most is conventional)of the 96 well plate.
- Fill the rest of the wells in the rows that you are using with 180 μL sterile water.
- Take the multi-channel pipettor, set it to 20 μL, pipette the cells in the first column of wells up and down a few times to suspend, take 20 μL from these wells and deposit it in the adjacent (to the right) column of cells. Pipette up and down to suspend.
- After you pipette to mix the cells, take 20 μL from this set of wells and do the same for the next. Continue until you reach the last column of wells on the plate.
- KEEP your container of ethanol AWAY from your flame!!! Sterilize the frogger by dipping the prongs into EtOH. After a quick shake insert the prongs into a Bunsen burner flame. Be CAREFUL not to burn yourself. KEEP the flames AWAY from your ethanol container so it doesn't catch on fire. Allow the frogger to cool slightly (1 minute). Alternatively, you can dip the frogger in sterile water for about 10 to 15 seconds to cool. Be sure to shake off the excess water. If you do not allow the frogger to cool enough, you will kill your cells and the frogging won't work well.
- Align the prongs of the frogger with the wells and then place the prongs into the wells. Wiggle the frogger within the well to ensure that the culture in each well is uniformly mixed. Stamp frogger firmly but gently onto an agar plate. Repeat this step to stamp additional plates.