The impact of fold-dilution and diluent volume on the estimation of colony forming units in a bacterial culture
Considering the non-uniform distribution of bacteria in a liquid culture, accurate enumeration of bacteria by serial dilution is important yet challenging in microbiological research. The deviation of estimated population size from the actual population size is anticipated in view of the sampling error arising at dilution, which is known to increase at each successive dilution; and the sampling error associated with the sample volume, where it is inversely proportional. In the dilution process of such population following a Poisson distribution, the relation of sampling error with the sequentially diluted suspensions is yet to be empirically demonstrated. Also, as a given-fold dilution can be achieved by passaging of either a higher or a lower sample volume, the sampling error associated in this case with the volume being passaged also remains to be evaluated. In view of that, the current experimental study was undertaken to further understand bacterial distribution and the underlying sampling error in dimensions previously not demonstrated. We demonstrated that sampling error in the enumeration process is independent of the bacterial cell densities of the diluted suspensions, which was on contrary to the theoretical assumptions. Moreover, sampling error is also not dependent on the number of passages in the dilution process. Importantly, we also demonstrated that passaging either a higher or lower sample volumes during serial dilution yielded similar enumeration, which was exceptional with regards to the statistical fluctuations inherent to a Poisson distribution.