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    Viability staining of Cryptosporidium oocysts and Giardia cysts combined with flow cytometry

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    Authors
    Schets FM
    Medema GJ
    Type
    Report
    Language
    en
    
    Metadata
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    Title
    Viability staining of Cryptosporidium oocysts and Giardia cysts combined with flow cytometry
    Translated Title
    Levensvatbaarheidskleuring van Cryptosporidium oocysten en Giardia cysten gecombineerd met flow cytometrie
    Publiekssamenvatting
    Flow cytometrie als extra zuiveringsstap heeft de detectiemethode voor Cryptosporidium oocysten en Giardia cysten in water verbeterd. Door middel van flow cytometrie kunnen (oo)cysten gescheiden worden van het meeste debris dat in waterconcentraten aanwezig is, waardoor de toepassing van fluorogene vitale kleurstoffen om de levensvatbaarheid van (oo)cysten vast te stellen mogelijk wordt. Kleuren met DAPI (4',6-dimidine-2-fenylindol-dihydrochloride) en propidiumjodide (PI), gevolgd door sorteren met de Becton Dickinson FACSort flow cytometer, resulteerde in microscopische preparaten waarin de afleesbaarheid van de levensvatbaarheidskleuring uitstekend was. PI positieve en PI negatieve oocysten werden evengoed gesorteerd en sorteren had geen effect op de levensvatbaarheid van de oocysten. Ook centrifugeren, sonificeren, floteren en wassen hadden geen effect. Dehydratie van membraanfilters, om toepassing van DIC microscopie mogelijk te maken, had een negatief effect op de levensvatbaarheid. Alle monster opwerk stappen veroorzaakten een aanzienlijk verlies in levensvatbaarheid bij Giardia cysten. De DAPI/PI kleuring geeft informatie over de levensvatbaarheid van Cryptosporidium oocysten, maar deze is onvolledig omdat DIC microscopie niet toegepast kan worden om te bepalen of DAPI en PI negatieve oocysten als dood of levend beschouwd moeten worden. De hier gebruikte detectiemethode kan slechts de aanwezigheid van Giardia cysten in monsters water aantonen en geeft geen informatie over de levensvatbaarheid van de gedetecteerde cysten omdat die door de methode beinvloed wordt. Andere methoden, zoals celkweek technieken, zijn waarschijnlijk meer geschikt om vast te stellen of (oo)cysten uit milieumonsters een risico vormen voor de volksgezondheid<br>
    The incorporation of flow cytometry as an additional purification step has improved the detection method for Cryptosporidium oocysts and Giardia cysts in water. Flow cytometry allows separation of (oo)cysts from interfering debris particles present in water concentrates and thus enables the application of fluorogenic vital dyes such as propidium iodide (PI) to assess (oo)cyst viability. Staining with DAPI (4',6-diamidine-2-phenylindole-dihydrochloride) and PI, followed by cell sorting with the Becton Dickinson FACSort flow cytometer, resulted in microscopic preparations in which the readability of vital staining was excellent. Sorting had no effect on oocyst viability and both PI positive and negative oocysts were sorted equally efficiently. Sample processing steps (centrifugation, sonification, flotation, washing) did not affect oocyst viability. Clarification of membrane filters on to which oocyst were sorted, by dehydration, in order to enable the use of DIC microscopy , had a strong negative effect on oocyst viability. All sample processing steps caused a considerable decrease in Giardia cyst viability, indicating that the current detection method can only detect the presence of Giardia cysts it cannot give information on their viability. Extension of the detection method with the DAPI/PI stain provides information on Cryptosporidium oocyst viability. The information is limited, since DIC microscopy cannot be used to examine whether DAPI and PI negative oocysts have contents and should be considered viable or dead. Due to the limitation of vital staining assays, other methods to assess oocyst viability are still needed. Cell culture techniques may prove valuable tools in the near future.<br>
    Publisher
    Rijksinstituut voor Volksgezondheid en Milieu RIVM
    Sponsors
    DGM/DWL
    URI
    http://hdl.handle.net/10029/255528
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