Abstract
This protocol was developed to utilize imaging flow cytometry (IFCM) in combination with fluorescent dyes to both enumerate and analyze morphological features of live and dead cells in a mixed live/dead bacterial sample. The fluorescent dyes used in this protocol include 5(6)-carboxyfluorescein diacetate (CFDA), which indicates the functional activity of esterase inside viable bacterial cells, and DRAQ7, a dye that exploits membrane-compromised bacterial cells to enter and stain the cell. The live cell population stained with CFDA emits a fluorescent green color while the dead cell population stained with DRAQ7 emits a fluorescent red color, which allows the two populations to be distinctively separated by the IFCM system. Additionally, the cytometer captures a clear image of each object, which can then be analyzed for morphology features. The IFCM system is able to reliably, accurately, and precisely determine a bacterial cell concentration as long as the concentration of cells in a sample is no less than 1 × 10(3) cells/ml. The two dyes, CFDA and DRAQ7, have been demonstrated to be an effective stain combination for bacterial viability analysis.
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