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Authors: Ken Stedman, Geoffrey Diemer, Jennifer E Kyle
Group: VERVE Net
Summary: An alternative epifluorescence microscopy (EFM) technique for virus enumeration has been developed using polycarbonate Track Etch (PCTE) filter membranes in place of aluminum oxide Anodisc membranes. The established EFM technique, first developed by Hennes and Suttle in 1995 and later expanded upon by Noble and Fuhrman in 1998, uses supported 20 nm pore-size Anodisc filter membranes to determine virus abundance in natural environments. Increased price and sporadic availability of Anodisc filters stimulated the evaluation of alternative filters for use in the procedure. The feasibility of using 30 nm pore-size PCTE filters for virus enumeration was assessed using the Anodisc filter procedure as a control. Although virion particle counts are slightly less precise using PCTE filters, they offer a substitute for Anodiscs while requiring only minor adjustments to the established protocol. Per slide, the PCTE costs approximately ten times less to prepare than the Anodisc-based method.

Proper citation: Ken Stedman, Geoffrey Diemer, Jennifer E Kyle 2016. PCTE Protocol for VLP enumeration. protocols.io https://dx.doi.org/10.17504/protocols.io.eaubaew Copy   

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Authors: André M. Comeau and Rachel T. Noble
Group: VERVE Net, Suttle Laboratory of Marine Molecular Microbiology and Virology
Summary: The steps describe how to prepare each virus concentrate. There are multiple options for many of the steps; in the case where there is more than one option they are noted in annotations.

Proper citation: André M. Comeau and Rachel T. Noble 2016. Concentration of viruses and preparation of FLVs for tracer assays. protocols.io https://dx.doi.org/10.17504/protocols.io.dzv765 Copy   

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Authors: Steven Wilhelm
Group: VERVE Net, The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), Samantha R Coy's Protocols
Summary: Adapted from: Van Etten, J. (n.d.). Titering of Chlorella Viruses. Retrieved from http://ncv.unl.edu/vanettenlab/

Proper citation: Steven Wilhelm 2017. Virus Purification by Sucrose Density Gradients. protocols.io https://dx.doi.org/10.17504/protocols.io.g4gbytw Copy   

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Authors: David Dunigan and Irina Agarkova
Group: VERVE Net

Proper citation: David Dunigan and Irina Agarkova 2016. DNA Gels. protocols.io https://dx.doi.org/10.17504/protocols.io.erubd6w Copy   

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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab

Proper citation: Matthew Sullivan 2016. Plating Prochlorococcus and Synechococcus strains in top agarose for plaque assays. protocols.io https://dx.doi.org/10.17504/protocols.io.c3vyn5 Copy   

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Authors: John H. Paul and Markus Weinbauer
Group: VERVE Net
Summary: These are protocols from: Paul, J. H., and M. Weinbauer. 2010. Detection of lysogeny in marine environments, p. 30–33. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO.Please see the published manuscript for additional information.

Proper citation: John H. Paul and Markus Weinbauer 2016. Detection of Lysogeny in Marine Environments. protocols.io https://dx.doi.org/10.17504/protocols.io.ebjbakn Copy   

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Authors: Howard Ochman
Group: VERVE Net, Sullivan Lab
Summary: Modified from H. Ochman protocol: Environ. Micro. 2009 Kunin, Engelbrekson Ochman & Hugenholtz

Proper citation: Howard Ochman 2016. 16s Pyrotag PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.c5iy4d Copy   

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Authors: K. Eric Wommack, Télesphore Sime-Ngando, Danielle M. Winget, Sanchita Jamindar, and Rebekah R. Helton
Group: VERVE Net
Summary: This protocol outlines and evaluates the filtration steps needed for the preparation of samples (viral concentrates) containing a density of viral particles concentrated from large volumes of natural water samples. (Fig. 2A and B)

Proper citation: K. Eric Wommack, Télesphore Sime-Ngando, Danielle M. Winget, Sanchita Jamindar, and Rebekah R. Helton 2015. General protocol for the concentration of virioplankton from a large volume (>20 L) water sample. protocols.io https://dx.doi.org/10.17504/protocols.io.dzq75v Copy   

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Authors: Joshua Weitz
Group: VERVE Net
Summary: BiMat: a MATLAB package to facilitate the analysis of bipartite networks. Extensive documentation, code, and worked examples available on: http://bimat.github.ioThe datasets included with the package include phage-bacterai infection networks. 

Proper citation: Joshua Weitz 2016. BiMat: a MATLAB package to facilitate the analysis of bipartite networks. protocols.io https://dx.doi.org/10.17504/protocols.io.fajbicn Copy   

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Authors: Bonnie Poulos, Jenn Brum, Elke Allers, Christine Schirmer
Group: VERVE Net, Sullivan Lab
Summary: This protocol consists of several methods for the preservation of seawater samples to later perform various techniques in the lab. It is meant as a sampling treatment in the field and is written as comprehensive; however, depending on needs and scientific questions, any or all of the methods may be used.Flow Cytometry (FCM): Samples are used for counting bacteria and for sorting different groups of microbes (eg, sorting synechococcus and prochlorococcus and non-fluorescent bacteria). Glutaraldehyde fixes microbes and renders them unculturable whereas microbes preserved in DMSO and Betaine can usually be cultured after thawing and rinsing. Betaine was developed for single cell sorting and may be a better preservative than DMSO.SYBR staining: Samples are used for counting bacteria (unfiltered or pre-filtered seawater) and viruses (unfiltered, pre-filtered or 0.2um filtered seawater) microscopically after staining with SYBR-Gold (or SYBR-Green). Transmission Electron Microscopy (TEM): Samples are used for determining frequency of visibly infected cells (FVIC) and determining types of microbes or viruses present, and can be quantitative.Quantitative PCR (qPCR): Samples are used for quantitative PCR of microbes. They can also be used for quantitative PCR of viruses.Viral qPCR: Samples are used for quantitative PCR of viruses.Culturing Viruses: Samples are used for cultivating viruses by solid or liquid plaque assay on susceptible hosts. Samples can also be used for viral tagging (using flow cytometry) and most probable number (MPN) assays.Fluorescence in situ hybridization (FISH): Samples are used with fluorescent probes to determine what populations of bacteria or other microbes are present. The filters may also be used to simply count the number of microbes in a sample. For detailed FISH procedure see here.

Proper citation: Bonnie Poulos, Jenn Brum, Elke Allers, Christine Schirmer 2015. Treatments for the Preservation of Seawater Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.c29yh5 Copy   

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Authors: Benjamin Bolduc
Group: VERVE Net, Sullivan Lab, iVirus
Summary: A collection of protocols designed to guide the user in processing a viral metagenome from raw sequence data to assembly, and subsequent analysis. The user uses actual reads from Ocean Sampling Day (2014) and processes them entirely within Cyverse, a NSF-supported cyberinfrastructure.

Proper citation: Benjamin Bolduc 2019. Applying vContact to Viral Sequences and Visualizing the Output (Cyverse). protocols.io https://dx.doi.org/10.17504/protocols.io.x5xfq7n Copy   

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Authors: Jonathan King
Group: VERVE Net, Sullivan Lab
Summary: This mixture is used in the CsCl Step Gradient to Purify Phage Protocol

Proper citation: Jonathan King 2016. CsCl Step Gradient Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.dmj44m Copy   

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Authors: B.R. Cunningham, J.R. Brum, S.M. Schwenck, M.B. Sullivan, S.G. John
Group: VERVE Net, Sullivan Lab
Summary: Purpose: This method for the enumeration of aquatic viruses is a low-cost alternative to the commonly used filter-mount method. Briefly, fluorescently-stained samples are wetmounted directly onto slides for epifluorescence microscopy after an optional chemical flocculation concentration step used for samples with anticipated virus concentrations of 7 viruses mL-1 (samples with >5×107 viruses mL-1 do not require this concentration step prior to analysis). Virus concentration in the wet-mounted sample is determined from the ratio of viruses to microsphere beads, which are added at a known concentration. This wet-mount method for enumerating viruses is significantly less expensive than the filter-mount method (i.e., the cost of microsphere beads per sample is ~500-fold lower than the cost of one filter per sample), and is appropriate for rapid, precise and accurate enumeration of aquatic viruses over a wide range of viral concentrations encountered in field and laboratory samples. The only limitation of this method is that samples with virus concentrations ≤1×106 viruses mL-1 cannot be enumerated, as the abundance of viruses is too low for efficient enumeration.Figure 1. Overview of the wet-mount method for enumeration of aquatic viruses.

Proper citation: B.R. Cunningham, J.R. Brum, S.M. Schwenck, M.B. Sullivan, S.G. John 2016. Wet-mount Method for Enumeration of Aquatic Viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.c8pzvm Copy   

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Authors: Corina Brussaard, Anne-Claire Baudoux, Anna A. M. Noordeloos, Marcel J. W. Veldhuis
Group: VERVE Net
Summary: An adapted dilution method was used to estimate viral lysis and microzooplankton grazing simultaneously. Parallel dilution experiments were performed with 30 kDa ultrafiltrate (virus and grazer-free diluent) and 0.2 µm filtered seawater (grazer-free, but virus-containing diluent). Specific viral lysis rates were calculated from the difference in P. globosa growth rates between the 2 dilution series after 24 h incubation under natural conditions. The validity of this method was tested using a culture of P. globosa infected with a known P. globosa virus (PgV).Protocol derived from:  Baudoux, A., Noordeloos, A., Veldhuis, M., & Brussaard, C. (2006). Virally induced mortality of Phaeocystis globosa during two spring blooms in temperate coastal waters. Aquat. Microb. Ecol. Aquatic Microbial Ecology,44(3), 207-217.

Proper citation: Corina Brussaard, Anne-Claire Baudoux, Anna A. M. Noordeloos, Marcel J. W. Veldhuis 2016. Simultaneous measurement of grazing and viral lysis of phytoplankton. protocols.io https://dx.doi.org/10.17504/protocols.io.dx67rd Copy   

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Authors: Jozef Nissimov
Group: VERVE Net, Bidle Lab
Summary: This general protocol can be used for assessing eukaryotic populations with various diagnostic stains (SYTOX for live/dead cells; DAF-FM for nitric oxide producing cells; DCFDA for reactive oxygen species; others). It is geared for samples that are ~5-10 µm in size, using a 70 µm orifice.

Proper citation: Jozef Nissimov 2015. Flow cytometry analysis of diatoms and coccolithophores with diagnostic stains (via a BD Biosciences Accuri C6). protocols.io https://dx.doi.org/10.17504/protocols.io.d9i94d Copy   

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Authors: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen
Group: VERVE Net
Summary: For use in "Isolation of cyanophages by liquid enrichment assay"

Proper citation: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen 2016. Propagation/Amplification of Lytic Agent. protocols.io https://dx.doi.org/10.17504/protocols.io.dq85zv Copy   

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Authors: Ross Fu and Matt Sullivan
Group: VERVE Net, Sullivan Lab
Summary: Purpose: to determine the concentration of DNA-containing particles in a given sample; useful in finding cell counts or phage titers.

Proper citation: Ross Fu and Matt Sullivan 2016. Protocol for SYBR Counts of Cyanophages and Bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.c7wzpd Copy   

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Authors: Larry Kane
Group: VERVE Net
Summary: This collection is part of the VERVE holiday drive to collect exotic off-the-shelf laboratory recipes. More details here.From Larry Kane: Back when I was a tech before grad school, made my own instant coffee by lyophilizing coffee (and drank it)(Image: Instant ! (Explored), Craig Sunter, CC BY-ND)

Proper citation: Larry Kane 2015. Kane Lab Lyophilized Coffee. protocols.io https://dx.doi.org/10.17504/protocols.io.ebabaie Copy   

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Authors: Elizabeth Silva
Group: VERVE Net
Summary: This protocol is part of the VERVE holiday drive to collect exotic off-the-shelf laboratory recipes. More details here.(Image: Frosty apple, Olga Filonenko, CC BY-SA)

Proper citation: Elizabeth Silva 2015. Ice-cold Effervescent Lab Apple. protocols.io https://dx.doi.org/10.17504/protocols.io.ebfbajn Copy   

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Authors: John H. Paul and Markus Weinbauer
Group: VERVE Net
Summary: This protocol gives a method for isolating temperate phages from marine viral concentrations. This protocol uses the conventional plaque agar overlay and looking for turbid or haloed plaques, a hallmark of temperate phages.Paul, J. H., and M. Weinbauer. 2010. Detection of lysogeny in marine environments, p. 30–33. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO.Please see the full chapter for additional details.

Proper citation: John H. Paul and Markus Weinbauer 2016. Isolation of temperate phages by plaque agar overlay. protocols.io https://dx.doi.org/10.17504/protocols.io.dpd5i5 Copy   

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