A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity

Abstract

The viability of Giardia muris cysts was studied with the fluorogenic dyes fluorescein diacetate (FDA) and propidium iodide (PI). G. muris cysts were seen to fluoresce intensely green with FDA at an excitation wavelength of 450 to 490 nm. Cysts stained with PI fluoresced bright orange at an excitation wavelength of 450 to 490 nm and bright red at 545 to 546 nm. Examination of isolated G. muris cyst preparations stained with FDA-PI revealed that greater than 85% of the cysts stained green with FDA and less than 15% stained orange-red with PI. Using the mouse model for giardiasis, we inoculated FDAor PI-stained cysts into neonatal mice. Feces were examined at days 3, 5, 8, and 11 postinoculation for the presence of cysts. Using 1,000 FDA-stained cysts as the inoculum, we detected cysts at days 5, 8, and 11 postinoculation in 19 of 19 mice, whereas a 50-fold greater dose of cysts produced infection in 27 of 27 mice at day 3 as well as at days 5, 8, and 11 postinoculation. Inoculation of mice with either 5,000 or 50,000 PI-stained G. muris cysts did not produce infection in any of the animals. Necropsy of mice infected with FDA-stained cysts showed trophozoites within the intestines. No trophozoites were detected within animals inoculated with PI-stained cysts. These results demonstrate that FDA-positive cysts are viable, as determined by infectivity, while PI-positive cysts are nonviable and incapable of producing G. muris infections in vivo. The development of this rapid and inexpensive test should facilitate testing of the effects of various chemical agents and environmental factors on the viability of Giardia cysts.

Applicability of the fluorescein diacetate assay for metabolic activity measurement of Microcystis aeruginosa (Chroococcales, Cyanobacteria)

“…It has been demonstrated that the desaturation of membrane lipids is essential to low temperature tolerance in cyanobacteria. The desaturation of fatty acids occurs without de novo synthesis of fatty acids during low temperature acclimation of cyanobacterial cells (Singh et al 2002) through acylase, which has been implicated in the hydrolysis of FDA (Schupp & Erlandsen 1987). Second, substances that include nucleophilic and basic amino acids, including histidine and cysteine, have been reported to induce abiotic cleavage of FDA (Clarke et al 2001;Wanandy et al 2005).…”

Giardia Cysts in Tertiary‐Treated Wastewater Effluents: Are They Infective?

“…Cyst viability may be assessed using differential interference contrast (DIC) microscopy and vital dye assays. The uptake of the vital dye, propidium iodide, by cysts has been shown to correlate with the inability to excyst and infect animals (Schupp and Erlandsen, 1987). Nonetheless, viability assessment based on vital dye inclusion or exclusion is considered only an estimate of infectivity.…”

Assessment of the Cell Viability of Cultured Perkinsus marinus (Perkinsea), a Parasitic Protozoan of the Eastern Oyster, Crassostrea virginica, Using SYBRgreen–Propidium Iodide Double Staining and Flow Cytometry

“…and Giardia spp. (Campbell, Robertson, and Smith 1992;Foster et al 2003;Kato and Bowman 2002;Medema et al 1998;Schupp and Erlandsen 1987;Vesey et al 1994; see also Quintero-Betancourt, Peele, and Rose 2002 for review), and of intra-erythrocytic protozoans, such as Plasmodium falciparum (Srivastava, Rottenberg, and Vaidya 1997), Babesia bovis (Wyatt, Goff, and Davis 1991), Leishmania amazonensis (Bertho, Cysne, and Coutinho 1992;Guinet et al 2000), and Theileria sergenti (Yagi et al 2000).…”