Lyndon L. Gyürék

Nucleic acid stains as indicators of Cryptosporidium parvum oocyst viability

We developed nucleic acid dye staining methodology for untreated, heat-treated and chemically inactivated C. parvum oocysts. The nucleic acid staining was compared to in vitro excystation and animal infectivity using split samples of oocysts. Among the nucleic acid stains tested, SYTO-9, hexidium and SYTO-59 stained the oocysts consistently, and the staining was related to the infectivity of the oocysts to neonatal CD-1 mice but not to in vitro excystation. The nucleic acid viability assay was used to determine log-inactivations of the oocysts after treatment with ozone, chlorine, chlorine dioxide and combinations of different chemical disinfectants, and was found to indicate log-inactivation levels similar to that of animal infectivity. A combined immunofluorescence-nucleic acid staining assay was developed for the oocysts of C. parvum and this assay will be invaluable for the detection and viability of oocysts in the laboratory and in environmental samples.

Comparison of Animal Infectivity and Nucleic Acid Staining for Assessment of Cryptosporidium parvum Viability in Water

ABSTRACT Cryptosporidium parvum oocysts were stained with the fluorogenic dyes SYTO-9 and SYTO-59 and sorted by flow cytometry in order to determine whether the fluorescent staining intensity correlated with the ability of oocysts to infect neonatal CD-1 mice. Oocysts that did not fluoresce or that displayed weak fluorescent intensity when stained with SYTO-9 or SYTO-59 readily established infections in mice, whereas those oocysts that fluoresced brightly did not. Although fluorescent staining profiles varied among different batches of oocysts, a relative cutoff in fluorescent staining intensity that correlated with animal infectivity was observed for all batches.

Nucleic acid stains as indicators of Giardia muris viability following cyst inactivation.

A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.

Comparison Between Animal Infectivity and Nucleic Acid Staining for Determination of Viability of Ozone-Inactivated Cryptosporidium parvum Oocysts

Abstract The viability of untreated and ozone treated Cryptosporidium parvum oocysts was determined using the nucleic acid dye SYTOO® staining evaluated by epi-fluorescence microscopy and flow cytometry. The use of microscopy to enumerate ozone treated oocysts, stained with SYTO-9, was found to either over- or underestimate the reduction in infectivity of parasites depending on the level of ozonation. Flow cytometry fluorescence histograms were generated for oocysts exposed to a range of ozone contact times. The median fluorescence intensity of ozone treated oocysts was found to be proportional to the reduction in mouse infectivity. A general linear regression model was fitted to the SYTO-9 staining data to give a predictive model for use with flow cytometry which provides a good estimate of the reduction in infectivity of C. parvum oocysts exposed to ozone in laboratory water.