title: Fluorescence decay of dyed protozoa: differences between stressed and non-stressed cysts.
abstract: Several series of tests have shown that fresh, intact samples of Giardia duodenalis and Cryptosporidium parvum (oo)cysts are not marked by fluorescent probes such as carboxyfluorcein-succinimidyl-diacetate-ester (CFDA-SE), C12-resazurin and SYTOX® Green, probably because of their robust cell walls. These dyes fail to indicate the viability of such protozoa and allow negative responses to be recorded from living and infectious samples. Cryptosporidium parvum showed stronger isolation from chemicals, with living oocysts remaining unstained by the probe for up to 90 days after extraction. However, in further fluorescence decay (FD) experiments run with G. duodenalis samples stained using CFDA-SE (comprising living, non-stressed but aged cysts, heat-killed samples and UV-C-stressed samples) each showed a different FD decay profile, here studied in seven series of tests of five replicates each. The FD profiles were fitted by double-exponential decay kinetics, with the decay constant k2 being five times higher than k1. This FD procedure is fast and can be easily reproduced in 10 steps, taking ~ 1 h of laboratory work for already purified samples.
authors.affiliation: School of Technology, University of Campinas/Limeira, SP, Brazil.|School of Agricultural Engineering, University of Campinas/Campinas, SP, Brazil.
authors.forename: Samuel Ricardo
authors.initials: SR
authors.lastname: dos Santos
authors.affiliation: Biology Institute, University of Campinas/Campinas, SP, Brazil.
authors.forename: Nilson
authors.initials: N
authors.lastname: Branco
authors.affiliation: Biology Institute, University of Campinas/Campinas, SP, Brazil.
authors.forename: Regina Maura Bueno
authors.initials: RM
authors.lastname: Franco
authors.affiliation: School of Agricultural Engineering, University of Campinas/Campinas, SP, Brazil.
authors.forename: José Euclides Stipp
authors.initials: JE
authors.lastname: Paterniani
authors.affiliation: Central Research Labs, Hamamatsu Photonics K.K., Hamamatsu City, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Katsumata
authors.affiliation: School of Biological Sciences, University of Bristol, UK.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Barlow
authors.affiliation: School of Technology, University of Campinas/Limeira, SP, Brazil.
authors.forename: Cristiano de Mello
authors.initials: Cde M
authors.lastname: Gallep
mesh_terms: D004396:Coloring Agents / Q000737:chemistry*; D016785:Cryptosporidium parvum / Q000737:chemistry*; D003560:Cysts / Q000737:chemistry*; D005453:Fluorescence; D016829:Giardia lamblia / Q000737:chemistry*
chemical_list: D004396:Coloring Agents
keywords: Cryptosporidium parvum; Giardia lamblia; carboxyfluorescein-diacetate-succinimidyl-ester; dyes; fluorescence decay