Public Lab Protocols from the Tucson Marine Phage Lab

Download protocol: Ferric_Chloride_Precipitation-TMPL_v6.pdf

by Seth G John, Carolina B Mendez, Li Deng, Bonnie Poulos, Anne-Kathryn Kauffman, Suzanne Kern, Jennifer Brum, Martin F Polz, Ed A Boyle, Matthew B Sullivan

"A simple and efficient method for concentration of ocean viruses by chemical flocculation", Environ Microbiol Rep. 2011 3(2):195-202

Ocean viruses alter ecosystems through host mortality, horizontal gene transfer and by facilitating remineralization of limiting nutrients. However, the study of wild viral populations is limited by inefficient and unreliable concentration techniques. Here, we develop a new technique to recover viruses from natural waters using iron-based flocculation and large-pore-size filtration, followed by resuspension of virus-containing precipitates in a pH 6 buffer. Recovered viruses are amenable to gene sequencing, and a variable proportion of phages, depending upon the phage, retain their infectivity when recovered. This Fe-based virus flocculation, filtration and resuspension method (FFR) is efficient (> 90% recovery), reliable, inexpensive and adaptable to many aspects of marine viral ecology and genomics research.

Download protocol: Concentrating_Viruses_with_Centrifugal_Ultrafiltration_Devices.pdf

This protocol describes how to concentrate viruses in liquid samples using an Amicon or Nanosep centrifugal ultrafiltration device. We use Amicons to concentrate medium volumes of samples (10s to 100s of mls) down to a final volume of ~4 ml. We use Nanoseps to concentrate smaller volumes of sample (<10 ml) down to a final volume of ~30 μl.

Download protocol: Linker_Amplification-TMPL_v5.pdf

by Melissa Duhaime, Li Deng, Amanda Borens, Bonnie Poulos, Matthew Sullivan

Here, we adapt the linker amplified shotgun library (LASL) approach to next generation sequencing by offering an alternate polymerase for challenging samples, developing a more efficient sizing step, integrating a “reconditioning PCR” step to increase yield and minimize late-cycle PCR artifacts, and empirically documenting the quantitative capability of the optimized method with both laboratory isolate and wild community viral DNA.

Our optimized linker amplification method requires as little as 1 pg of DNA and is the most precise and accurate available, with amplification biases less than 1.5-fold, even for complex samples as diverse as a wild virus community. While optimized here for 454 sequencing, this linker amplification method can be used to prepare metagenomics libraries for sequencing with next-generation platforms, including Illumina and Ion Torrent.

Download protocol: FASP_Protocol_Viruses.pdf
Download protocol: General_2D_LCMSMS_Method.pdf

Collaborator, Nathan VerBerkmoes (Oak Ridge National Labs), worked with us to develop more sensitive proteomics assays for viral isolates which we are now also using for environmental viral concentrates. A new sample prep method (FASP) was optimized to maximize the signal from our commonly low-protein containing samples, and then run using 2d-LC-MS-MS to maximize detection across the large dynamic range in isolate and environmental samples. Most of the protocol development presented here was funded by a Gordon and Betty Moore Foundation grant to MBS.

Download protocol: Adsorbing_Viruses_onto_TEM_Grids.pdf

This protocol describes how to adsorb viruses onto TEM (transmission electron microscopy) grids. The sample is allowed to sit on a hydrophilic grid and viruses adsorb onto the surface of the grid. This technique is generally used for viral lysates with high concentrations of viruses. For natural samples, use the protocol "Quantitatively Depositing Viruses onto TEM Grids using an Airfuge".

Download protocol: Quantitatively_Depositing_Viruses_onto_a_TEM_Grid_using_an_Airfuge.pdf

by Jennifer R. Brum and Grieg F. Steward

Brum, J.R., and Steward, G.F. (2010) Morphological characterization of viruses in the stratified water column of alkaline, hypersaline Mono Lake. Microbial Ecology 60: 636-643.

This protocol describes how to quantitatively deposit viruses onto TEM grids using an Airfuge (an air-driven ultracentrifuge made by Beckman). The purpose of using this protocol is to prepare aquatic samples to obtain viral assemblage characteristics (e.g. morphotypes, capsid widths, tail lengths) using transmission electron microscopy (TEM).

Download protocol: Positive_and_Negative_Stainging_of_Viruses_on_TEM_Grids.pdf

This protocol describes how to negatively or positively stain viruses that have been deposited onto TEM grids so that they can be imaged using transmission electron microscopy (TEM). Positive staining stains the virus itself such that the virus is dark against a lighter background (Figure 1A). This is the easiest staining to do and yields results suitable for analyzing the morphological diversity of viruses from aquatic samples. Negative staining relies on some stain remaining around the edges of a virus such that the details of the virus are clearly defined (Figure 1B). Negative staining is more difficult and is used to obtain publication quality micrographs of viruses.

Download protocol: FVIC_Protocol.pdf

This protocol describes how to pellet bacterial cells onto grids and stain them so that viruses can be visualized within the cells using transmission electron microscopy. Data obtained from these grids can be used to calculate the frequency of visibly infected cells (FVIC), the frequency of infected cells (FIC), the fraction of mortality due to viral lysis (FMVL), and burst size.

Download protocol: Analysis_of_Viral_Morphological_Characteristics.pdf

This protocol describes how to analyze a natural aquatic virus sample using transmission electron microscopy (TEM). The purpose is to obtain the capsid diameter distributions of the viral assemblage, tail length distributions of the viral assemblage, and percentages of each viral morphotype in a sample.

Download protocol: Using_ImageJ_to_Measure_Viral_Dimensions_in_Micrographs.pdf

This protocol describes the use of ImageJ to measure dimensions of viruses in TEM micrographs, but can be applied to the measurement of anything in any image.