Gel Microdroplets: Rapid Detection and Enumeration of Individual Microorganisms by their Metabolic Activity

Abstract

We report a new, flexible method for rapid detection and enumeration of individual microorganisms using small (e.g. 10 to 100 micron diameter) gel particles surrounded by a non-aqueous liquid with low dielectric constant. Primary samples without prior cultivation can be used. In the present study, gel microdroplets (GMDs) surrounded by an inert oil were statistically inoculated such that GMDs had a high probability of initially containing either zero or one acid-producing microorganism. Such GMDs retained dissociable metabolites produced by individual cells (or microcolonies) within the small GMD volume. The accumulated metabolic acids led to rapid changes in pH within GMDs initially occupied by one microorganism or colony forming unit (CFU), while GMDs with zero microorganisms had unchanged pH. The cumulative activity within individual GMDs was then determined using pH sensitive fluorescence indicators. This method was used to enumerate individual cell viability directly, without any prior culture, from clinically infected urine samples in about 1.5 hours for several rapidly growing pathogens, and was in agreement with much slower conventional culture methods. Because GMDs can be made readily in large numbers, and because many indicator systems can be used, GMDs used with automated measurement apparatus should have wide applicability.