I'm challenging bacteria with different compounds, then plating the suspension on LB agar to assess CFU and understand the bactericidal properties of my compounds. However, I'm encountering significant CFU variability between replicates of the same conditions and even controls. For example, in a recent experiment, I obtained counts of 57, 31, and 9 on three different LB plates where the bacteria were only exposed to PBS. I'm vortexing the bacterial suspension before adding it to the compounds and trying to be as uniform and consistent as possible with everything, but still experiencing a lot of variability. Any tips would be highly appreciated!
Are you vortexing your samples/dilutions right before plating? They will settle to the bottom of tubes over time, even when shaken.
But honestly, having less than 1 log of variability between replicates of the same condition when plating bacteria for CFUs isn't that uncommon or concerning. You'll almost never end up with something as neat as "50, 53 and 49 colonies" from three replicates. Typically you're looking for >1 log differences to say anything unless you repeat the experiment a lot of times.
I concur with Alexandra that this degree of variation is pretty normal. To some degree you can get better data if you have more colonies on a plate. I see a lot more consistency when you have around 100 colonies or so.
You'll want either plate out a higher volume or suspend in a lower volume to get a useful number of colonies. Your cells are way too diluted.
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