I am quantifying CFU/mL of two bacterial subpopulations at various time points. However, I am encountering a significant issue: at a given dilution and using the same plating volume, the number of colonies on replicate plates is showing a large discrepancy. For instance, two plates from the same dilution yielded 19 and 87 colonies, respectively.
I am aware that some variability is expected in microbiological techniques, but this degree of difference seems unusually high. Could this be due to uneven mixing before plating, clumping of cells, pipetting inconsistencies, or something else entirely?
I would appreciate any insights or suggestions to minimize such variation.
The cell count is normal, if it was 19 and >300 then it would be an issue. Just give a quick vortex before you make each serial dilution, and before plating. That would ensure the samples are properly mixed. It's best to make three plate replicates.
Hello Swati Sharma , there can be many reasons for the problem you are facing. A few reasons which you mentioned can be the culprit, so here are a few things you can try to minimise this.
1. Always vortex or invert dilution tubes vigorously just before every aliquot is taken.
2. If it's a clumping issue, you can try adding a non-ionic detergent like Tween-80 (0.001 to 0.005%) if it doesn't affect the viability.
3. To reduce the pipetting errors, use properly calibrated pipettes and avoid frothing of the sample. You can also increase the plating volume to minimise errors.
4. One of the reasons can be improper and uneven spreading on the agar plate.
Hopefully, this will help.
Hello Swati Sharma , this issue can be resolved by proper mixing of the culture before every step of serial dilution. Since the CFU/ml were calculated at various time points (according to the procedure mentioned in the question), it is very important that all the replicates have same number cells before the start of the experiment (for e.g. normalizing the culture to 0.1 OD600 ). Even little discrepancies in the normalizing the culture can lead to huge differences as the time increases. The CFU/ml at initial time points might not show huge differences in the replicates but as the time progresses significant changes in the CFU count might be observed.
Like Simant Kumar and Pavlos Trus have mentioned the other reasons could be improper mixing, uneven spreading and Pipetting errors.
Hope this would answer the question.
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