Flow Cytometry and GMDs

GMD encapsulation allows the analysis of microcolony growth using flow cytometry, facilitating rapid and automated evaluation of microbial growth or stasis in response to anti-microbial agents. Flow cytometry has not been routinely used as an analytical tool for microbiology in part because of the tendency of microorganisms such as fungi and bacteria to aggregate and because of the variability of viability staining. Use of the GMD Growth Assay to analyze samples could significantly shorten the culture time required for analysis of drug susceptibility. GMD encapsulation also permits sorting and isolation of sub-populations of interest such, as drug resistant cells, for subsequent evaluation including gene sequence determination.

Flow Cytometric Quantification of Growth of Yeast in GMDs

The above data demonstrate that growth of microorganisms such as yeast can be quantified in GMDs using flow cytometry. C. albicans 64545 was encapsulated, stained with SYTO 13, and analyzed using a Coulter Epics Elite flow cytometer equipped with a 488 nm laser. The left panels are dot plots showing log of forward versus 90° angle light scatter and the right panels are the corresponding histograms showing SYTO 13 fluorescence intensity of encapsulated C. albicans 64545 at times 0 hr, 1.0 hr, 2.0 hr, and 3.0 hr. Histograms were gated on R1 which contain occupied GMDs. Unoccupied GMDs which have less forward-angle scatter appear to the left of R1. The SYTO 13 signal is typically 400-fold greater than the background fluorescence of the unoccupied GMDs used as a control. Mean fluorescence intensity doubled approximately every 1.5 hr, indicating the yeast doubling time.

© 1999, One Cell Systems, Inc.