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Applied and Environmental Microbiology, November 2005, p. 7321-7326, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7321-7326.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Catalyzed Reported Deposition-Fluorescence In Situ Hybridization Protocol To Evaluate Phagotrophy in Mixotrophic Protists

Juan M. Medina-Sánchez,1* Marisol Felip,2 and Emilio O. Casamayor1

Unitat de Limnologia, Departament de Biogeoquimica Aquàtica. Centre d'Estudis Avançats de Blanes-CSIC, E-17300 Blanes, Spain,1 Departament d'Ecologia i Centre de Recerca d'Alta Muntanya, Universitat de Barcelona, E-08028 Barcelona, Spain2

Received 4 April 2005/ Accepted 10 June 2005

We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists.

* Corresponding author. Mailing address: Unitat de Limnologia, Departament de Biogeoquimica Aquàtica, Centre d'Estudis Avançats de Blanes-CSIC, C/Accés a la Cala St. Francesc, 14, E-17300 Blanes (Girona), Spain. Phone: 34-972-336-101. Fax: 34-972-337-806. E-mail: jmmedina{at}ceab.csic.es.

Applied and Environmental Microbiology, November 2005, p. 7321-7326, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7321-7326.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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Copyright © 2005 by the American Society for Microbiology. All rights reserved.