title: Counting viruses and bacteria in photosynthetic microbial mats.
abstract: Viral abundances in benthic environments are the highest found in aquatic systems. Photosynthetic microbial mats represent benthic environments with high microbial activity and possibly high viral densities, yet viral abundances have not been examined in such systems. Existing extraction procedures typically used in benthic viral ecology were applied to the complex matrix of microbial mats but were found to inefficiently extract viruses. Here, we present a method for extraction and quantification of viruses from photosynthetic microbial mats using epifluorescence microscopy (EFM) and flow cytometry (FCM). A combination of EDTA addition, probe sonication, and enzyme treatment applied to a glutaraldehyde-fixed sample resulted in a substantially higher viral (5- to 33-fold) extraction efficiency and reduced background noise compared to previously published methods. Using this method, it was found that in general, intertidal photosynthetic microbial mats harbor very high viral abundances (2.8 × 10(10) ± 0.3 × 10(10) g(-1)) compared with benthic habitats (10(7) to 10(9) g(-1)). This procedure also showed 4.5- and 4-fold-increased efficacies of extraction of viruses and bacteria, respectively, from intertidal sediments, allowing a single method to be used for the microbial mat and underlying sediment.
authors.affiliation: Department of Biological Oceanography, Royal Netherlands Institute for Sea Research (NIOZ), Den Burg, The Netherlands Section for Marine Biology, University of Copenhagen, Helsingør, Denmark ccd.carreira@gmail.com.
authors.forename: Cátia
authors.initials: C
authors.lastname: Carreira
authors.affiliation: Section for Marine Biology, University of Copenhagen, Helsingør, Denmark.
authors.forename: Marc
authors.initials: M
authors.lastname: Staal
authors.affiliation: Section for Marine Biology, University of Copenhagen, Helsingør, Denmark.
authors.forename: Mathias
authors.initials: M
authors.lastname: Middelboe
authors.affiliation: Department of Biological Oceanography, Royal Netherlands Institute for Sea Research (NIOZ), Den Burg, The Netherlands Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Corina P D
authors.initials: CP
authors.lastname: Brussaard
mesh_terms: D000498:Allyl Compounds; D001419:Bacteria / Q000302:isolation & purification*; D058491:Bacterial Load / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D019015:Geologic Sediments / Q000382:microbiology* / Q000821:virology*; D008856:Microscopy, Fluorescence / Q000379:methods; D013440:Sulfides; D019562:Viral Load / Q000379:methods*; D014780:Viruses / Q000302:isolation & purification*
chemical_list: D000498:Allyl Compounds; D013440:Sulfides; C028526:methylallyl trisulfide