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Label-Free Darkfield-Based Technique to Assist in the Detection of Giardia Cysts

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Abstract

Giardia is a pathogenic protozoan that has caused giardiasis outbreaks worldwide, and this water-borne disease may occur due to faults in water supply and treatment systems. To support surveillance and control programs, the monitoring of this parasite is essential in water samples. Although Giardia cyst detection is usually based on standard light microscopy, the resulting low contrasted cysts together with a wide array of particles of comparable size to the target protozoan demands a high level of observer skill and experience, as well as a long time to process the individual samples. The visualization of this protozoan can be significantly improved by using staining procedures, e.g., Lugol’s iodine in brightfield or fluorescence-based methods such as fluorescence-isothiocyanate (FITC) and 4′,6-diamidino-2-phenylindole (DAPI). However, the significant costs make fluorescence microscopy difficult to be performed in less-developed countries. Accordingly, the present study evaluated the effectiveness of inexpensive darkfield (DF) setups to acquire higher contrasted images of unstained cyst samples as a direct and rapid method for monitoring protozoa. Two low-cost custom-made DF illumination modes, transmitted and reflected, were evaluated on a standard light microscope. Examination of purified Giardia cyst suspensions with both DF setups revealed a direct correlation between morphological appearance and uptake of DAPI. Images captured under transmitted illumination showed higher contrast and sharpness when compared to the reflected images. DF microscopy might provide a simple, direct, and inexpensive method for observing Giardia cysts, which shows basic aspects of their intracellular structure, although the applicability of the method to raw water concentrates remains to be demonstrated.

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Funding

This work was supported by the São Paulo Research Foundation (FAPESP-Brazil) under Grant Processes 2014/12712-8 and 2012/50522-0 and by the Coordination for the Improvement of Higher Education Personnel (CAPES-Brazil) for the Master’s scholarship awarded to Bárbara Luíza Souza Freitas.

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Authors and Affiliations

  1. Department of Electrical Engineering, Federal University of São Carlos, Rodovia Washington Luís, km 235, São Paulo, 13565-905, Brazil

    Valdinei Luís Belini

  2. Department of Hydraulics and Sanitation, São Carlos School of Engineering, University of São Paulo, Trabalhador São-Carlense Avenue, 400, São Paulo, 13566-590, Brazil

    Bárbara Luíza Souza Freitas & Lyda Patricia Sabogal-Paz

  3. Protozoology Laboratory, Biology Institute, University of Campinas, University City Zeferino Vaz, São Paulo, Campinas, 13083-970, Brazil

    Nilson Branco & Regina Maura Bueno Franco

Authors
  1. Valdinei Luís Belini
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  2. Bárbara Luíza Souza Freitas
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  3. Lyda Patricia Sabogal-Paz
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  4. Nilson Branco
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  5. Regina Maura Bueno Franco
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Correspondence to Valdinei Luís Belini.

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Belini, V.L., Freitas, B.L.S., Sabogal-Paz, L.P. et al. Label-Free Darkfield-Based Technique to Assist in the Detection of Giardia Cysts. Water Air Soil Pollut 229, 195 (2018). https://doi.org/10.1007/s11270-018-3834-x

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  • DOI: https://doi.org/10.1007/s11270-018-3834-x

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