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Fluorescence decay of dyed protozoa: differences between stressed and non-stressed cysts
Samuel Ricardo dos Santos1,2, Nilson Branco3, Regina Maura Bueno Franco3
1School of Technology, University of Campinas/Limeira, SP, Brazil.
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.
Insights
Common fluorescent probes fail to detect viable Giardia duodenalis and Cryptosporidium parvum (oo)cysts due to their robust cell walls. A new fluorescence decay (FD) method offers a rapid and reproducible way to assess protozoan viability.
Area of Science:
- Parasitology
- Microbiology
- Biotechnology
Background:
- Giardia duodenalis and Cryptosporidium parvum are significant protozoan pathogens.
- Accurate viability assessment of protozoan cysts is crucial for water quality monitoring and treatment efficacy.
- Current viability assays using fluorescent probes are unreliable for these protozoa due to their resistant cell walls.
Purpose of the Study:
- To evaluate the efficacy of common fluorescent probes for Giardia duodenalis and Cryptosporidium parvum viability assessment.
- To develop and validate a novel fluorescence decay (FD) method for rapid and reliable viability determination of these protozoa.
Main Methods:
- Testing of fresh, intact Giardia duodenalis and Cryptosporidium parvum (oo)cysts with fluorescent probes (CFDA-SE, C12-resazurin, SYTOX® Green).
- Investigation of Cryptosporidium parvum oocyst stability in isolation for up to 90 days.
- Application of fluorescence decay (FD) experiments on Giardia duodenalis samples (living, aged, heat-killed, UV-C-stressed) stained with CFDA-SE.
- Analysis of FD profiles using double-exponential decay kinetics.
Main Results:
- Standard fluorescent probes failed to stain fresh, intact Giardia duodenalis and Cryptosporidium parvum (oo)cysts, yielding false-negative viability results.
- Cryptosporidium parvum oocysts remained unstained for up to 90 days, indicating probe impermeability.
- Distinct fluorescence decay profiles were observed for different states of Giardia duodenalis cysts.
- FD profiles were accurately fitted by double-exponential decay kinetics, with k2 five times higher than k1.
Conclusions:
- Common fluorescent viability probes are unsuitable for Giardia duodenalis and Cryptosporidium parvum.
- The developed fluorescence decay (FD) method provides a fast, reproducible, and accurate approach for assessing the viability of these protozoan cysts.
- This FD method has potential applications in water safety and public health.
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