Accurately measuring the number of viable microorganisms plays an essential role in microbiological studies. Since the conventional agar method of enumerating visible colonies is time-consuming and not accurate, efforts have been made towards overcoming these limitations by counting the invisible micro-colonies. However, none of studies on micro-colony counting was able to save significant time or provide accurate results. Herein, we developed an on-glass-slide cell culture device that enables rapid formation of micro-colonies on a 0.38 mm-thick gel film without suffering from nutrient and oxygen deprivation during bacteria culturing. Employing a phase contrast imaging setup, we achieved rapid microscopic scanning of micro-colonies within a large sample area on the thin film without the need of fluorescent staining. Using Escherichia coli (E. coli) as a demonstration, our technique was able to shorten the culturing time to within 5 h and automatically enumerate the micro-colonies from the phase contrast images. Moreover, this method delivered more accurate counts than the conventional visible colony counting methods. Due to these advantages, this imaging-based micro-colony enumeration technique provides a new platform for the quantification of viable microorganisms.
Keywords: digital; enumeration; micro-colonies; microscopic scanning; viable microorganism.