Rapid microbial detection and enumeration using gel microdroplets and colorimetric or fluorescence indicator systems.

A new micromethod employing gel microdroplets (GMDs) and optical measurements can be used for rapid detection and enumeration of viable microorganisms (J. C. Weaver, G. B. Williams, A. M. Klibanov, and A. L. Demain, Bio/Technology 6:1084-1089, 1988) and has several potential applications in clinical microbiology. This method involves entrapping microorganisms in GMDs (10 to 100 microns in diameter) which are surrounded by a hydrophobic (low dielectric) fluid, subsequently distinguishing occupied and unoccupied GMDs with colorimetric or fluorescence indicators, counting both occupied and unoccupied GMDs, and applying Poisson statistical analysis. Acid-producing microorganisms were used to compare colorimetric and fluorescence pH indicator systems. Fluorescence systems were generally superior, particularly for detection before microbial growth occurred. Although colorimetric detection was reasonably fast for fast-growing microorganisms, significantly longer times were needed for slow-growing microorganisms. We investigated the dependence of the detection time on microbial division time, GMD size, and buffering capacity of the medium within GMDs. It was found possible to use a single preparation of GMDs, containing a range of GMD sizes, to simultaneously provide a viable enumeration of growing and nongrowing (e.g., stressed) cells. This was possible because small GMDs responded rapidly to both growing and nongrowing cells, while large GMDs, although slower, responded much more rapidly to growing cells than to nongrowing cells. Separate analysis of small and large GMDs in the same preparation yielded two enumerations, one of nongrowing cells and the other of growing cells. GMDs can also be used with conventional light microscopy to detect and enumerate fast-growing acid-producing bacteria much more quickly than conventional plating methods.