Counting Viruses and Bacteria in Photosynthetic Microbial Mats

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 x 10(10) +/- 0.3 x 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.