The impact of fold-dilution and diluent volume on the estimation of colony forming units in a bacterial culture

Accurate enumeration of bacteria in a culture is the first step in both fundamental as well as applied research in microbiology. Serial dilution is an age old method used widely by researchers for enumerating viable bacteria in a culture where a specific sample volume is passaged successively to a specific diluent volume. Here, we demonstrated that a higher sample volume is a better representation of bacterial population than a lower sample volume, which was in concordance with the random nature of bacterial distribution in culture. Therefore, a bigger sample to diluent ratio during serial dilution appears more favorable for an accurate bacterial enumeration than a smaller ratio. But surprisingly, enumeration using the different dilution ratios such as 1:9, 1:99 and 1:999 in 1.0 mL final volume yielded similar results with the exception of 1:999, where 1 L sample was passaged. However, in 10.0 mL final volume of dilution, the above three dilution ratios exhibited similar bacterial enumeration. The experiment was performed using two different bacterial cultures such as Escherichia coli and Ralstonia pseudosolanacearum. Our results indicated that the advantage gained due to lesser number of passages in case of a lower sample volume could overcome the disadvantage associated with it, thereby co-aligning the different dilution ratios with regards to enumeration. Hence, although in laboratory, 1:9 dilution ratio is usually performed during serial dilution, our results suggest that dilution ratios such as 1:99 in 1 mL dilution volume and ratios such as 1:99 and 1:999 in 10 mL dilution volume are equally effective, which also reduces time, cost and labor.