Checking a basic technique

A conversation with the new post-doc, and then with the RA, raised questions about how each of us does the serial dilutions and platings we use to measure cell numbers. These measurements are so fundamental to our research that we haven't questioned whether we're all doing them right.

• Do we always use a pipettor and disposable tips, or do we sometimes use glass pipettes?
• Do we use relatively large volumes, in culture tubes, or small volumes in microfuge tubes?
  
• Do we always make 1-in-10 dilutions, or sometimes make 1-in-100 or other proportions?
• When we use a pipettor, do we use a fresh pipette tip every time, or do we only change tips when we think it matters? Every time we sample from a different tube? Only if the new tube has a higher concentration of cells? Only if the new tube has a lower concentration of cells? Only if the volume we need to measure changes? What about when we're using glass pipettes - do we use a fresh one every time?
  
• Do we pipette liquid up and up and down in the tip or pipette before removing our sample? Do we pipette liquid up and down to rinse the tip or pipette out after putting the sample into the new tube?
  
• Do we always plate the same volume of dilution onto each agar plate, or do we use different volumes to refine our measurements?

This afternoon we're going to do a test, to find out whether these differences matter. I've grown cultures of two E. coli strains overnight, one wildtype and one resistant to kanamycin. And I've poured lots of plates, with and without kanamycin. I'm going to mix a bit of the KanR culture into the wildtype culture, and then we're all going to dilute and plate the mixed culture to estimate the density of wildtype and KanR cells.

If we all get the same answers, we'll know that our differences in technique don't matter. But if we get different answers then we'll need to investigate further.