Determination of the viability of Trichomonas vaginalis using flow cytometry.
In clinical laboratories, viability of Trichomonas vaginalis is determined by using light microscopy (differential count of motile to nonmotile organisms). Alternative methods are proposed that utilise flow cytometry. Under an epifluorescence microscope, live organisms fluorescence intensely green with fluorescein diacetate (FDA), whereas dead cells fluoresce orange with propidium iodide (PI). Flow cytometric histograms of green versus red fluorescence reveal distinct populations for live and dead cells. The anionic oxonal probe DiBAC4(3) is a membrane potential sensitive dye that distributes between the inside of the cell and the medium. Live organisms are less fluorescent than dead organisms when stained with the oxonol probe. Valinomycin, dicyclohexylcarbodiimide, and vanadate all give significant changes in the fluorescence intensities of cultures stained with the oxonol probe compared with control cultures, indicating that this probe is detecting changes in plasma membrane potential. Both FDA/PI and oxonol staining protocols allow good discrimination between populations and permit counts that are more statistically significant than those obtained by light microscopy. These methods remove the subjectiveness of microscopic counts and would increase the accuracy of susceptibility assays.
Microbiology Group, School of Pure and Applied Biology, University of Wales College of Cardiff, U.K.
M J
MJ
Humphreys
R
R
Allman
D
D
Lloyd
D000818:Animals; D001463:Barbiturates; D002470:Cell Survival / Q000187:drug effects; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D007555:Isoxazoles; D008564:Membrane Potentials; D014246:Trichomonas vaginalis / Q000166:cytology* / Q000187:drug effects; D014634:Valinomycin / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D001463:Barbiturates; D005456:Fluorescent Dyes; D007555:Isoxazoles; C052795:bis(1,3-dibutylbarbiturate)trimethine oxonol; D014634:Valinomycin; D014638:Vanadates; D004024:Dicyclohexylcarbodiimide