Fluorescence decay of dyed protozoa: differences between stressed and non-stressed cysts Samuel Ricardo dos Santos, a,b Nilson Branco, c Regina Maura Bueno Franco, c José Euclides Stipp Paterniani, b Masakazu Katsumata, d Peter W. Barlow e and Cristiano de Mello Gallep a * 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 k 2 being five times higher than k 1 . This FD procedure is fast and can be easily reproduced in 10 steps, taking ~ 1 h of laboratory work for already purified samples. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: fluorescence decay; Giardia lamblia; Cryptosporidium parvum; carboxyfluorescein-diacetate-succinimidyl-ester; dyes Introduction Giardia lamblia (syn. G. duodenalis and G. intestinalis) and Crypto- sporidium parvum are challenging and important organisms in modern environmental monitoring. These protozoa can seriously affect humans and animals, and reflect sanitation problems in water quality control, with consequently huge impacts on eco- nomics and public health (1–3). Standard protocols, including EPA method 1623.1 (4) and AWWA 9711 (5), are used to detect Giardia and Cryptosporidium, although they are not able to distinguish between living and dead (oo)cysts. Techniques that use the excystation of (oo)cysts and the inclusion or exclusion of vital dyes such 4′,6-diamidino-2- phenylindole (DAPI) and propidium iodide (PI) are used to detect cyst viability. However, studies using these methods have given conflicting results, indicating that the probes may overes- timate the viability of (oo)cysts (6–9). The DAPI probe is able to properly assess only the morphological properties, enabling better image capture, and is used as a confirmatory step in combination with the EPA 1623.1 method (5,9,10). Infectivity tests remain the best tool by which to define the real risk of environmental contamination by parasitic protozoa. Currently, the procedure involves incubation in an animal model and fecal analysis after the protozoal life cycle has been com- pleted, to estimate the level of infectivity (11). However, this proce- dure is very expensive and may take a long time to give conclusive data. Moreover, most water-treatment stations are not able to maintain the animal models and sophisticated laboratory facilities needed to perform such infectivity tests and so guarantee that parasites have been killed properly. Research with dye probes aims to find useful, reliable and low- cost procedures for the detection of protozoa viability. The use of dyes in viability tests has been demonstrated for C. parvum (12) and G. lamblia (13). In addition, redox dyes, such as 5-cyano- 2,3-ditolyl tetrazolium chloride (CTC) and p-iodonitrotetrazolium (INT), have been used as probes to test for respiratory activity in G. lamblia and C. parvum (oo)cysts (14). Such a scanning procedure would be easier and quicker than the host incubation tests described above. Schupp and Erlandsen (15) tested the use of fluorescein diacetate (FDA) as a staining probe to detect the viability of Giardia muris. Using samples containing 1000 and 50,000 FDA-stained cysts, the authors detected infectivity in mice 5 days after inoculation, and so concluded that the stained cysts were alive (15). However, it is possible that a great number of unstained but viable cysts were also present in the samples and, hence, be the cause of the infection, a * Correspondence to: C. de Mello Gallep, School of Technology, University of Campinas/Limeira, SP, Brazil. E-mail: gallep@ft.unicamp.br a School of Technology, University of Campinas/Limeira, SP, Brazil b School of Agricultural Engineering, University of Campinas/Campinas, SP, Brazil c Biology Institute, University of Campinas/Campinas, SP, Brazil d Central Research Labs, Hamamatsu Photonics K.K., Hamamatsu City, Japan e School of Biological Sciences, University of Bristol, UK Luminescence 2015 Copyright © 2015 John Wiley & Sons, Ltd. Research article Received: 30 October 2014, Revised: 27 December 2014, Accepted: 5 January 2015 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/bio.2872