Microliter spotting and micro-colony observation: a rapid and simple approach for counting bacterial colony forming units

Abstract

For enumerating viable bacteria, traditional dilution plating to count colony forming units (CFU) has always been the preferred method in microbiology owing to its simplicity, albeit laborious and time-consuming. Similar CFU counts can be obtained by quantifying growing microcolonies in conjunction with the perks of a microscope. Here, we employed a simple method of five microliter spotting of differently diluted bacterial culture multiples times on a single petri plate followed by finding out CFU by counting microcolonies using phase contrast microscope. In this method within four-hour period CFU of an Escherichia coli culture can be found out. Further, within ten-hour period, CFU in a culture of Ralstonia solanacearum, a bacterium with generation time around 3 h, can be estimated. The CFU number determined by microcolonies observed is comparable with that obtained by the dilution plating method. Microcolonies number 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 h, though there was noticeable increase in size of the colonies. It suggested that microcolonies observed in the early hours indeed represent the bacterial number in the culture. Though manual, the method is less labor intensive apart from being simple, easy, economical and rapid.

Generation of green electricity from sludge using photo-stimulated bacterial consortium as a sustainable technology

“…For quantitative analysis, sludge samples were vortexed for 3 mins to be completely homogenous, one mL of each sample was suspended separately in 9 mL of sterile saline, a series of decimal dilutions were prepared, 0.1 mL of each sample was plated on nutrient agar medium and incubated for 24 h at 37°C, total viable count (TVC) was expressed as colony forming units (cfu/mL) of the sample, experiments were undertaken in triplicates [21].…”

A study on twitching motility dynamics in Ralstonia solanacearum microcolonies by live imaging

“…Although various studies reported about the twitching motility inR. solanacearum and the factors associated with it (Kai et al, 2015;Kang et al, 2002;Ray et al, 2015;Singh et al, 2018), its impact on the microcolony shape and size (Bhuyan et al 2023) in this bacterium in a density-dependent manner has not been reported yet. More importantly, the dynamics as well as the directionality of twitching motility exhibited by peripheral cells in a colony are yet to be studied adequately.…”

A study on twitching motility dynamics in Ralstonia solanacearum microcolonies by live imaging

“…Although various studies reported about the twitching motility in R. solanacearum and the factors associated with it [13–15, 20], its impact on the microcolony shape [21] in this bacterium in a density‐dependent manner has not been reported yet. More importantly, the dynamics as well as the directionality of twitching motility exhibited by peripheral cells in a colony are yet to be studied adequately.…”