Once more with Resolution
Before you head off, I just want to say a few words about resolution.
Even if you have perfect technique in mixing and pipetting, you cannot determine the exact number of bacteria present in your sample. This is an unavoidable consequence of diluting and scaling up.
Let's imagine that we have a sample with exactly 183,000 cells per mL. And let's say that our technique is flawless. If we count a 1:10,000 diluted plate, we should find 183 CFUs (remember, we have magically flawless technique). But the exact same thing would happen if our sample started with 183,001 cells. Or 183,100 cells. Or 183,400 cells. Or 182,800 cells. In fact, even with our absolutely flawless technique, the best we can say is that there are between 182,500 and 183,500 cells. So our absolutely unavoidable error is plus or minus 500/182,000 -- about 0.27%. That's pretty good.
Let's look at some other ways we could count the same 183,000 cell sample:
| Dilution Factor | plate count | resolution | ease of counting |
|---|---|---|---|
| 1:1000 | 1830 | +/- 50 0.027% |
NOPE, can't do it |
| 1:10,000 | 183 | +/- 500 0.27% |
boring but do-able |
| 1:100,000 | 18 | +/- 5000 2.7% |
easy |
| 1:1,000,000 | 2 | +/- 50,000 27% |
chimpanzees can do this |
So you can see that the 25 to 250 guideline is really a compromise between the ability to distinguish non-overlapping CFUs, and the resolution afforded by the scaling up component.
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