Rapid microbial detection and enumeration using gel microdroplets
and colorimetric or fluorescence indicator systems.
Williams GB; Weaver JC; Demain AL J Clin Microbiol (HSH), 1990 May; 28 (5): 1002-8
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.