{Reference Type}: Journal Article
{Title}: Microliter spotting and micro-colony observation: A rapid and simple approach for counting bacterial colony forming units.
{Author}: Bhuyan S;Yadav M;Giri SJ;Begum S;Das S;Phukan A;Priyadarshani P;Sarkar S;Jayswal A;Kabyashree K;Kumar A;Mandal M;Ray SK;
{Journal}: J Microbiol Methods
{Volume}: 207
{Issue}: 0
{Year}: 04 2023 15
{Factor}: 2.622
{DOI}: 10.1016/j.mimet.2023.106707
{Abstract}: For enumerating viable bacteria, traditional dilution plating to count colony forming units (CFUs) has always been the preferred method in microbiology owing to its simplicity, albeit being laborious and time-consuming. Similar CFU counts can be obtained by quantifying growing micro-colonies in conjunction with the benefits of a microscope. Here, we employed a simple method of five to ten microliter spotting of a diluted bacterial culture multiple times on a single Petri dish followed by determining CFU by counting micro-colonies using a phase-contrast microscope. In this method, the CFU of an Escherichia coli culture can be estimated within a four-hour period after spotting. Further, within a ten-hour period after spotting, CFU in a culture of Ralstonia solanacearum, a bacterium with a generation time of around 2 h, can be estimated. The CFU number determined by micro-colonies observed for 106-fold dilutions or lower is similar to that obtained by the dilution plating method for 107-fold dilutions or lower. Micro-colony numbers observed in the early hours of growth (2 h in case of E. coli and 8 h in case of R. solanacearum) were found to remain consistent at later hours (4 h in case of E. coli and 10 h in case of R. solanacearum), where the visibility of the colonies was better due to a noticeable increase in the size of the colonies. This suggested that micro-colonies observed in the early hours indeed represent the bacterial number in the culture. Practical applications to this counting method were employed in studying the rifampicin-resistant mutation rate as well as performing a fluctuation test in E. coli. The spotting method described here to enumerate bacterial CFU results in reduction of labour, time and resources.