No abstract

A two-color assay employing competing surface mAbs reduces the number of fluorescent particles sorted, thus improving FCM detection methods for Cryptosporidium.

We report the first demonstration of directly recording fluorescence spectra of single cells in flow cytometry. An intensified, 512-element photodiode array was used in conjunction with a dispersing prism to capture the fluorescence emission spectra of Coulter ImmunoCheck calibration beads and Dictyostelium discoideum spores stained with the indocarbocyanine derivative CY3, fluorescein isothiocyanate, or R-phycoerythrin. The demonstration was made feasible by enhancing the signal-to-noise ratio of the detection process by using a fast gating technique applied to the detector. Results show that the complete fluorescence spectra of individual stained cells contain information that is normally not captured by conventional flow cytometers. By using the spectrographic flow cytometer, all this information is recorded, allowing small features and shifts in the fluorescence spectra of labelled particles to be studied.

Fluorescent labelling methods for detecting microorganisms in water have limited sensitivity partly due to the natural autofluorescence from environmental particles. The aim of this study was to examine the autofluorescence of water samples to determine the optimal excitation source and fluorescent labels for minimising background autofluorescence and therefore enhancing sensitive detection of Cryptosporidium oocysts. Particles concentrated from water were examined using fluorimetry at a wide range of excitation wavelengths to determine their autofluorescent properties. Two major peaks were identified emitting at 390 to 510 nm and at 640 to 700 nm. Flow cytometry was used to define the optical properties of oocysts immunofluorescently labelled with a range of fluorochromes. Concentrated water samples were analysed using flow cytometry and the number of particles with fluorescence and light scatter properties similar to the fluorescently labelled oocysts recorded. Fluorescein isothiocyanate exited at 488 nm was the most suitable label for oocysts in untreated water with less than 70 particles having optical properties similar to labelled oocysts, detected in 10 litre concentrates. The fluorochromes CY3, phycoerythrin (PE), and tetramethylrhodamine B thioisocyanate (TRITC) excited at 542 nm were the most suitable labels for oocysts in drinking water with less than 40 particles having optical properties similar to labelled oocysts, detected in 100 litre concentrates. Cytometry 29:147–154, 1997. © 1997 Wiley‐Liss, Inc.

A variety of methods have been used to enumerate Cryptosporidium parvum oocysts from source or drinking waters. The reliability of these counting methods varies, in part, with suspension density, sample purity, and other factors. Frequently, the method of determination of suspension density is not reported by authors. To confound the problem, each method of counting has large inherent variation. There is a relationship between suspension density, overall number of organisms counted, and counting mechanism accuracy that should be accounted for when selecting a counting mechanism. This study selected a maximum acceptable coefficient of variation (CV) to be 10%. A method was considered unreliable if this standard was not achieved. Flow cytometry achieved this standard at 486 oocysts/ml. Counting with a Coulter counter achieved this level of reliability at about 1,230 oocysts/ml. Neither chamber slides nor fluorescent antibody-stained well slides ever demonstrated less than 10% CV. However, estimates of the minimum required concentrations were 5,100 oocysts/ml and approximately 6,500 oocysts/ml, respectively. The hemacytometer provided counts accurate to a 10% CV at a concentration of at least 60,000 organisms/ml. Of the methods tested, flow cytometry provided the least amount of variability at low suspension densities.