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.

Parasiticidal effect of synthetic bovine lactoferrin peptides on the enteric parasiteGiardia intestinalis

“…Consequently, PI is an indirect indicator of cellular viability. On the other hand, it has been established that an intact lipid bilayer slows the leakage of the fluorochrome CFDA from within intact cells, while injured or stressed cells cannot retain or accumulate the fluorochrome CFDA (dos Santos et al 2015;Schupp et al 1988). Additionally, the replication time is different in the distinct genotypes of G. intestinalis; consequently, the drug sensitivity data from in vitro studies in Giardia varies as a function of the replication time and the methodological techniques employed (Hahn et al 2013).…”

“…The vials were incubated at 37°C for 12 h, chilled on ice to detach trophozoites, and centrifuged at 500 g for 10 min, and the pellet was resuspended in 1 ml of medium. Long-term viability was determined using the fluorescent probe carboxyfluorescein-succinimidyl-diacetate-ester D r a f t (CFDA-SE) (10 µg/ml) and visualized by epi-fluorescense microscopy (dos Santos et al 2015). Experiments were performed at least three times in triplicate, and the mean and standard deviations are indicated.…”