Fluorescence decay of dyed protozoa: differences between stressed and non-stressed cysts

Author(s)

dos Santos, Samuel Ricardo ^{[1, 2]} ; Branco, Nilson ^[3] ; Bueno Franco, Regina Maura ^[3] ; Stipp Paterniani, Jose Euclides ^[1] ; Katsumata, Masakazu ^[4] ; Barlow, Peter W. ^[5] ; Gallep, Cristiano de Mello ^[2]

Affiliation

^[1] Univ Estadual Campinas, Sch Agr Engn, Campinas, SP - Brazil

^[2] Univ Estadual Campinas, Sch Technol, Limeira, SP - Brazil

^[3] Univ Estadual Campinas, Inst Biol, Campinas, SP - Brazil

^[4] Hamamatsu Photon KK, Cent Res Labs, Hamamatsu, Shizuoka - Japan

^[5] Univ Bristol, Sch Biol Sci, Bristol BS8 1TH, Avon - England

• Document type: Journal article
• Source: LUMINESCENCE; v. 30, n. 7, p. 1139-1147, NOV 2015.
• Web of Science Citations: 1

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 (R) 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 similar to 1 h of laboratory work for already purified samples. Copyright (C) 2015 John Wiley \& Sons, Ltd. (AU)

• FAPESP's process: 10/07792-1 - ULTRAWEAK LIGHT EMISSION DETECTION IN Giardia spp
• Grantee: Samuel Ricardo dos Santos
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 11/10763-6 - Biophotonics applied to parasitology: measuring ultra-weak light emission from Cryptosporidium spp. and Giardia spp.
• Grantee: Cristiano de Mello Gallep
• Support Opportunities: Regular Research Grants
• FAPESP's process: 13/01501-3 - Ultra-weak light emission in protozoa
• Grantee: Samuel Ricardo dos Santos
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate