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Biotechniques. 2015 Apr 1;58(4):181-8. doi: 10.2144/000114274. eCollection 2015 Apr.

Transmission electron microscopy sample preparation protocols for the ultrastructural study of cysts of free-living protozoa.

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Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Unit of Coordination in Veterinary Diagnostics-Epidemiology and Risk Assessment, CODA-CERVA, Ukkel, Belgium.
Nematology Research Unit, Department of Biology, Ghent University, Ghent, Belgium.
Laboratory of Protistology and Aquatic Ecology, Department of Biology, Ghent University, Ghent, Belgium.


Cysts of free-living protozoa have an impact on the ecology and epidemiology of bacteria because they may act as a transmission vector or shelter the bacteria against hash environmental conditions. Detection and localization of intracystic bacteria and examination of the en- and excystment dynamics is a major challenge because no detailed protocols for ultrastructural analysis of cysts are currently available. Transmission electron microscopy (TEM) is ideally suited for those analyses; however, conventional TEM protocols are not satisfactory for cysts of free-living protozoa. Here we report on the design and testing of four protocols for TEM sample preparation of cysts. Two protocols, one based on chemical fixation in coated well plates and one on high-pressure freezing, were selected as the most effective for TEM-based ultrastructural studies of cysts. Our protocols will enable improved analysis of cyst structure and a better understanding of bacterial survival mechanisms in cysts.


intracellular survival; protozoan cysts; transmission electron microscopy; ultrastructural analysis

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