Viral enumeration using cost-effective wet-mount epifluorescence microscopy for aquatic ecosystems and modern microbialites

Abstract

ABSTRACT Enumeration is a fundamental measure of community ecology in which viruses represent the most numerous biological identities. Epifluorescence microscopy (EFM) has been the gold standard method for environmental viral enumeration for over 25 years. Currently, standard EFM methods using the Anodisc filters are no longer cost-effective (>$15 per slide) and have yet to be applied to modern microbialites. Microbialites are microbially driven benthic organosedimentary deposits that have been present for most of Earth’s history. We present a cost-effective method for environmental viral enumeration from aquatic samples, microbial mats, and exopolymeric substances (EPSs) within modern microbialites using EFM. Our integrated approach, which includes filtration, differential centrifugation, chloroform treatment, glutaraldehyde fixation, benzonase nuclease treatment, probe sonication (EPS and mat only), SYBR Gold staining, wet mounting, and imaging, provides a robust method for modern microbialites and aquatic samples. Viral abundances of modern microbialites and aquatic samples collected from Fayetteville Green Lake (FGL) and Great Salt Lake (GSL) did not differ across ecosystems by sample type. EPS and microbial mat samples had an order of magnitude higher viral-like particle abundance when compared to water regardless of the ecosystem (10 7 vs 10 6 ). Viral enumeration allows for estimates of total viral numbers and weights. The entire weight of all the viruses in FGL and GSL are ~598 g and ~2.2 kg, respectively. Further development of EFM methods and software is needed for viral enumeration. Our method provides a robust and cost-effective (~$0.75 per sample) viral enumeration within modern microbialites and aquatic ecosystems. IMPORTANCE Low-cost and robust viral enumeration is a critical first step toward understanding the global virome. Our method is a deep drive integration providing a window into viral dark matter within aquatic ecosystems. We enumerated the viruses within Green Lake and Great Salt Lake microbialites, EPS, and water column. The entire weight of all the viruses in Green Lake and Great Salt Lake are ~598 g and ~2.2 kg, respectively.