Name: Environmental Sampling of Photosynthetic Microbes and Their Viruses: From Field to Lab (Video) | JoVE Journal
Uploaded: 2025-07-03T14:30:00.000+00:00
Duration: 8 min 1 s
Description: Discover environmental sampling methods for cyanophages and their cyanobacterial hosts using electron microscopy, quantitative PCR, and high-throughput sequencing. This workflow enables isolation of single genotype virus stocks and characterization of virus-host dynamics for predictive virology models.

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5 minutes

This study was funded in part by the U.S. National Science Foundation (NSF) OISE grant no. 1856091 (PI-Ceballos) and NSF DBI grant no. 2119968 (PI-Ceballos). The authors acknowledge contributions of Dr. Chinnapong Wangnai and Dr. Chakrit Tachaapaikoon from King Mongkut&#39;s University of Technology Thonburi (Thailand), who provided videography and field support for environmental sampling, respectively. The authors also thank Dr. Jorge Armando Leiva Sanabria from the University of Costa Rica - Guanacaste for his assistance in securing sampling permits from National Parks in Costa Rica and assisting in sample collection from the geothermal pools. The authors likewise thank Mr. Enrique Marroquin, Jr. (University of California Merced) and Ms. Alexia Maceda (University of Maine, Orono, ME, USA), who assisted with sampling off the coast of southern California and lakes in northern Minnesota, respectively. The authors thank and acknowledge the technical support of Ms. Socheata Hour, who calculated infection parameters for the &#966;SBL14-SWII&#160;dataset.

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</ol>

The authors declare no conflicts of interest.

The virosphere is vast and diverse, thus requiring different experimental approaches for studying different virus systems. This includes different strategies for isolating viruses and their hosts from different environments and different processing methods to ensure that pure cultures of hosts and their viruses can be produced from raw samples. These pure cultures can be used in SVSH infection assays, both on solid media and in liquid culture, so that system features and quantitative analyses of virus-host dynamics can b...

Characterizing infection dynamics and evolutionary relationships of cyanophages and their hosts is key to understanding the current and future states of marine and freshwater ecosystems. Advanced high-throughput sequencing techniques and modern analytical methods allow rapid sequencing of cyanophage (and host) genomes to study virus-host relationships1,2. However, workflows begin with environmental sampling to isolate and identify viruses and their hosts3,4,5. Due to the diversity of the virosphere, there is no single protocol for sampling and characterizing all viruses and bacteriophages. Indeed, each virus-host system has its unique characteristics. Nonetheless, the baseline objectives are similar regardless of the virus system being explored: isolate the virus; determine host range; demonstrate productive infection in one or more hosts; characterize the virus, host, and virus-host infection dynamics using qualitative and quantitative approaches. An advanced understanding of the virus system can be examined by perturbing the system. Changing parameters such as multiplicity of infection (MOI)6, temperature, pH, the host strain, or by the application of drugs to determine how the system responds, provides additional insight into the nature of virus-host dynamics. More advanced manipulation, such as directed evolution, can provide additional information about the potential evolutionary trajectory of the system and how co-evolutionary endpoints may impact the larger microbial community and ecosystem.
In this study, we present a model (but readily adaptable) protocol for environmental sampling of photosynthetic microbes such as cyanobacteria and microalgae, which play a critical role in nutrient cycling and primary production in marine, freshwater, and other aquatic ecosystems7,8,9,10,11. The workflow focuses on isolating hosts from the euphotic zones of marine and freshwater environments and their viruses and bacteriophages, which are key drivers in these ecosystems12. Both viruses and bacteriophages impact both eukaryotic and prokaryotic population structures and heavily influence ecosystem diversity and productivity13. Thus, the goal here is to provide a sampling protocol and post-sampling workflow that facilitates enrichment of hosts and viruses for investigating: single-virus/single-host, multi-virus/single-host, single-virus/multi-host, and multi-virus/multi-host dynamics; -omics substrates underlying virus-host infection dynamics; and, potential co-evolutionary trajectories and evolutionary endpoints that impact the virus-host system under study and the larger ecosystem from which the viruses and host were extracted.
Specifically, we describe a workflow for environmental sampling, field pre-processing of samples (i.e., before reaching the lab), post-processing for isolation of bacteriophage (and viruses), enrichment of mixed-species cultures of microorganisms towards identifying permissive hosts, developing single-colony isolates of putative hosts, confirming a bona fide virus-host relationship (i.e., productive infection), and characterizing fundamental infection properties (e.g., virulence). Infection assays not only permit calculation of relative virulence (VR)14 and host resilience (RR)15 but also include steps for extracting culture samples at key time-points for investigating -omics substrates that underlie the observed physiology (i.e., the virus-host interaction dynamics). Lastly, we discuss potential pitfalls, troubleshooting, and solutions, as well as ways to modify select steps to target specific taxa. The workflow is applicable to cyanophage-cyanobacteria, phycodnavirus-microalgae, and thermophilic fusellovirus-archaeal systems16,17, demonstrating its utility in characterizing viruses across all three domains of life: Bacteria, Eukarya, and Archaea.

<table><tbody><tr><td>1 hp stainless steel transfer pump&amp;nbsp;</td><td>Utilitech, Charlotte, NC, USA</td><td>N/A</td><td></td></tr><tr><td>1.5 ml tubes</td><td>www.eppendorf.com</td><td>30123611</td><td></td></tr><tr><td>10K NMWL Centricon Plus 70 spin-concentrator&amp;nbsp;</td><td>www.sigmaaldrich.com</td><td>UFC701008</td><td></td></tr><tr><td>250 mL baffled Erlenmeyer flasks&amp;nbsp;</td><td>www.sigmaaldrich.com</td><td>CLS4450250</td><td></td></tr><tr><td>3800-Watt Inverter Generator&amp;nbsp;</td><td>WEN Products, West Dundee, IL, USA</td><td>WEN 56380i</td><td></td></tr><tr><td>3K NMWL Amicon 15 mL&amp;nbsp;</td><td>www.sigmaaldrich.com</td><td>UFC800308</td><td></td></tr><tr><td>500 mL baffled Erlenmeyer flasks&amp;nbsp;</td><td>www.sigmaaldrich.com</td><td>CLS4450500</td><td></td></tr><tr><td>5-gallon water jugs&amp;nbsp;</td><td>www.homedepot.com</td><td>N/A</td><td></td></tr><tr><td>80ft long, 0.5&amp;rdquo; diameter, nylon&amp;nbsp;</td><td>https://facomex.mx/producto/combo/</td><td>N/A</td><td></td></tr><tr><td>A 26 ft. deep-V center console Sailfish 2660 with two Yamaha 150 hp outboard motors&amp;nbsp;</td><td>https://yamahaoutboards.com/</td><td>N/A</td><td></td></tr><tr><td>Ammonium acetate (CH3CO2NH4)</td><td>www.sigmaaldrich.com</td><td>631618</td><td></td></tr><tr><td>Boric acid (H3BO3)</td><td>www.sigmaaldrich.com</td><td>10043353</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl2. 2H2O)</td><td>www.sigmaaldrich.com</td><td>10035048</td><td></td></tr><tr><td>Centrifuge 5920&amp;nbsp;R</td><td>www.eppendorf.com</td><td>5948000107</td><td></td></tr><tr><td>Citric Acid (C6H8O7)</td><td>www.sigmaaldrich.com</td><td>77929</td><td></td></tr><tr><td>Cobalt(II) nitrate hexahydrate [Co(NO3)2.6H2O]</td><td>www.sigmaaldrich.com</td><td>10026229</td><td></td></tr><tr><td>Collapse-resistant rubber flex hose&amp;nbsp;</td><td>swanhose.com</td><td>CELTF58050</td><td></td></tr><tr><td>Copper(II) sulfate pentahydrate (CuSO4.5H2O)</td><td>www.sigmaaldrich.com</td><td>7758998</td><td></td></tr><tr><td>Corning 50 mL centrifuge tubes&amp;nbsp;</td><td>www.sigmaaldrich.com</td><td>CLS430291</td><td></td></tr><tr><td>Dipotassium phosphate (K2HPO4)</td><td>www.sigmaaldrich.com</td><td>7758114</td><td></td></tr><tr><td>Disodium ethylenediaminetetraacetic acid (Na2EDTA)</td><td>www.sigmaaldrich.com</td><td>6381926</td><td></td></tr><tr><td>DS-11 FX+_Microvolume and Spectrophotometer and Florometer</td><td>www.denovix.com</td><td>N/A</td><td></td></tr><tr><td>Duct tape&amp;nbsp;</td><td>www.homedepot.com</td><td>N/A</td><td></td></tr><tr><td>Eppendorf tubes</td><td>www.sigmaaldrich.com</td><td>EP022364120</td><td></td></tr><tr><td>Ethanol</td><td>www.sigmaaldrich.com</td><td>64175</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>www.sigmaaldrich.com</td><td>60004</td><td></td></tr><tr><td>Ferric ammonium citrate (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;8&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;middot;xFe&lt;sup&gt;3+&lt;/sup&gt;&amp;middot;yNH&lt;sub&gt;3&lt;/sub&gt;)</td><td>www.sigmaaldrich.com</td><td>1185575</td><td></td></tr><tr><td>Flex-plus blue PVC tubing&amp;nbsp;</td><td>www.carlonsales.com</td><td>12008-750</td><td></td></tr><tr><td>Innova S44i - Stackable Incubator Shaker</td><td>www.eppendorf.com</td><td>2231001081</td><td></td></tr><tr><td>LVEM5 Benchtop Electron Microscope_Transmission electron microscopy (TEM)</td><td>delongamerica.com</td><td>N/A</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate (MgSO4.7 H2O)</td><td>www.sigmaaldrich.com</td><td>10034998</td><td></td></tr><tr><td>Manganese(II) chloride tetrahydrate (MnCl2.4H2O)</td><td>www.sigmaaldrich.com</td><td>13446349</td><td></td></tr><tr><td>Mushroom Anchor black Vinyl Coated/30 lbs</td><td>www.homedepot.com</td><td>N/A</td><td></td></tr><tr><td>Petri dishes, polystyrene</td><td>www.sigmaaldrich.com</td><td>P5481</td><td></td></tr><tr><td>Polyethersulfone (PES) membrane filter cup with 0.22 mm</td><td>www.sigmaaldrich.com</td><td>Z358193-1CS</td><td></td></tr><tr><td>Polyethersulfone (PES) membrane filter cup with 0.45 mm</td><td>www.sigmaaldrich.com</td><td>Z370622-1CS</td><td></td></tr><tr><td>Potassium ethyl xanthogenate (C2H5OCSSK)</td><td>www.sigmaaldrich.com</td><td>140896</td><td></td></tr><tr><td>Sodium carbonate (Na2CO3)</td><td>www.sigmaaldrich.com</td><td>497198</td><td></td></tr><tr><td>Sodium dodecyl sulfate [CH3(CH2)11OSO3Na]</td><td>www.sigmaaldrich.com</td><td>151213</td><td></td></tr><tr><td>Sodium molybdate dihydrate (Na2MoO4.2H2O)</td><td>www.sigmaaldrich.com</td><td>10102406</td><td></td></tr><tr><td>Sodium nitrate (NaNO3)</td><td>www.sigmaaldrich.com</td><td>7631994</td><td></td></tr><tr><td>Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic aci (TES) (C6H15NO6S)</td><td>www.sigmaaldrich.com</td><td>7365448</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>www.sigmaaldrich.com</td><td>T8787</td><td></td></tr><tr><td>Trizma hydrochloride (Tris-HCl)</td><td>www.sigmaaldrich.com</td><td>1185531</td><td></td></tr><tr><td>Vacuum/pressure pump&amp;nbsp;</td><td>Cole-Parmer; Vernon Hills, IL, USA</td><td>EW-79204-00</td><td></td></tr><tr><td>Vinyl tubing&amp;nbsp;</td><td>www.homedepot.com</td><td>N/A</td><td></td></tr><tr><td>Zinc sulfate heptahydrate (ZnSO4.7H2O)</td><td>www.sigmaaldrich.com</td><td>7446200</td><td></td></tr></tbody></table>

environmental sampling of photosynthetic microbes and their viruses: from field to lab

Environmental Sampling of Photosynthetic Microbes and Their Viruses: From Field to Lab

Environmental sampling of photosynthetic microorganisms and their viruses plays a critical role in understanding contemporary marine and freshwater biodiversity and ecosystem dynamics as well as the impacts of climate change-related factors (e.g., rising temperatures and acidification) on evolutionary trajectories of species and community composition. Unfortunately, the diversity of the virosphere does not support a single universal sampling and experimental workflow. Indeed, each virus system has unique features, which require modifications to standard protocols in virology to accomplish research goals. Although virus discovery and characterization require approaches that are specific to the target system, for all viruses, the research aims are similar: isolate the virus; determine host range; confirm productive infection; and characterize the virus, the host, and virus-host dynamics. Robust descriptions of virus-host systems consist minimally of elucidating morphology, physiology, biochemistry, and omics profiles. Further information may be obtained by manipulating the system by changing factors such as multiplicity of infection, temperature, pH, host-switch, directed evolution, or applying drugs to observe virus-host system response. Our laboratory studies viruses across domains of life (Archaea, Bacteria, and Eukarya). In this report, we detail methods for sampling photosynthetic microbes from the euphotic zone of freshwater and marine environments with focus on isolating bacteriophage (i.e., cyanophage) of cyanobacteria. Cyanobacteria are keystone species critical to primary production and nutrient cycling in these aquatic ecosystems. The described workflow extends from sampling waters at different depths to characterizing virus-host system features using liquid and solid media culture, advanced molecular/genetic methods, and analytical approaches. The methods described are adaptable to bacteriophage and virus discovery in virus-host systems across domains of life.

The use of a portable gasoline-powered electric generator, a water transfer pump, and collapse-resistant tubing serves as an effective way to extract water samples from the ocean, lakes, frozen lakes, and other aquatic environments (e.g., lagoons) at select depths (Figure 1, top three rows). Alternative equipment is required for high-temperature acid waters of geothermal hot springs, pools, and mud pots, which can feature pH &lt; 4 and temperatures greater than 90 °C. A long bamboo pole or t...

Here, we describe methods of sampling from the euphotic zones of marine and freshwater ecosystems to isolate cyanophages (and their hosts) and post-sampling processes that enrich single genotypes and characterize virus-host interaction dynamics.

Environmental sampling of photosynthetic microorganisms and their viruses plays a critical role in understanding contemporary marine and freshwater ...

The goal of these projects is to quantify physiological parameters in viral infections, then map omics substrates to the observed physiology, leading to models or laws in virology that predict changes in virus-host dynamics. With advances in high-throughput sequencing and streamlined bioinformatics workflows, multi-omics analyses are readily accessible, which provides an opportunity to explain physiological phenomena as a function of specific omics substrates. Using experimental infections, electron microscopy, quantitative PCR next-generation sequencing, and robust bioinformatics pipelines, network analyses can identify relationships between omics and physiological variables which inform the development of predictive models.The biggest challenge is generating pure single genotype virus stocks with accurate titers so that multiplicity of infection can be determined for experimental infections. Titer determination remains an issue in virology. We've developed metrics to calculate relative virulence between two viruses infecting a host or one virus infecting different hosts in single-host/single-virus infections.We've standardized a workflow that works across virus systems. To begin, perform environmental sampling offshore along the Southern California coastline. Attach a 10-foot-long collapse-resistant rubber flex hose to the outlet of the pump. Plug the transfer pump electrical cord into the inverter generator. Once assembled, prime the transfer pump and test to ensure the pump system is operational. Secure one end of a 25-meter-long nylon marine rope, having a diameter of 1.25 centimeters, to the eyelet of a 14-kilogram mushroom anchor.Use duct tape to secure the rope to the blue PVC tubing every three meters. Once a steady flow of water is being transferred, discharge one to two tube volumes back into the ocean to ensure that water from the desired depth is flowing through the pump. Collect water samples in 20-liter water jugs to transport them to the field station.Filter the water sample through a filter cup assembly, then connect the filter cup to a portable air diaphragm vacuum pump using clear vinyl tubing. Transfer the sample into the filter cup and begin filtration. When complete, transfer the filtrate into a water collection bottle and label it.Now use a scalpel to cut out the filter from the filter cup assembly. Cut it in half and transfer into a 50-milliliter centrifuge tube. Transfer 50 milliliters of the filtered sea water into the same centrifuge tube.To concentrate mixed samples, for each sampling depth by site, ship back the ultrafiltrate with a cell debris-laden 0.45 micrometer filter to the laboratory and centrifuge. Transfer 60 milliliters of the filtrate into a spin filter. Centrifuge it at 6, 000 g for 10 to 20 minutes to concentrate it.Repeat centrifugation until a 1.5 to 2 milliliter concentrate of the virus or bacteriophage suspension is obtained. Transfer the resultant concentrate into a two-milliliter Eppendorf tube for further transmission electron microscopy imaging of viruses or bacteriophage from the processed environmental sample. Streak environmental samples onto prepared agar plates.To isolate single-colony isolates of putative virus or bacteriophage hosts, use a sterile inoculation loop to transfer an isolate to a new plate. Then use the same loop to pick another colony and transfer it into a conical flask containing liquid broth. Prepare 10 seed cultures in 20-milliliter culture tubes containing five milliliters of BG-11 media.Add five milliliters of a mix of ultrafiltrate from the sample site and BG-11. Pick a single colony isolate from a culture plate with a sterile toothpick, then inoculate each tube individually by dropping one toothpick into each tube. Once positive seed culture growth is detected, repeat streak procedures until only a single colony phenotype appears on plates.Once cycles of liquid culture and solid media plate work result in a single cell phenotype, characterize microbial morphotypes using light microscopy. To detect virus activity, grow putative host lawns of cyanobacteria or microalgae from working stocks on BG-11 agar plates. Once individual plaques appear on plates from spotting on or swirling on virus suspension, use a sterile loop to scrape a single plaque.Swirl the loop in a 10-milliliter seed culture of the host. To determine host range, prepare lawns of multiple putative hosts isolated from environmental samples or obtained from other laboratories or culture collections. Generate host growth curves by taking optical density at 730 nanometer readings at regular time intervals from infected and uninfected cultures.Pure cultures of photosynthetic microorganisms displayed distinct morphologies under light microscopy. Based on morphological features and 16S rRNA sequencing followed by phylogenetic analysis, the isolate was identified as being a species of cyanobacteria from the family, Merismopediaceae. An alternative isolate showing oval-shaped green cells is suggestive of Synechococcus.The Synechocytis like strain SWII form dense, uniform lawns on BG-11 agar, ideal for plate-based plaque assays. phiSBL-14 infection caused visible plaque clearing in swirl-on lawn serial dilution plaque assays and impeded the growth of the strain SWII in liquid culture. Growth curves showed that SWII infected with concentrated phiSBL-14 suspension had a much slower increase in optical density compared to both the diluted phage suspension and the uninfected control.Maximum specific growth rate was highest in uninfected SWII cultures. Transmission electron microscopy of infected SWII cultures revealed cytopathic effects including membrane thickening, internal granules, and membrane blebbing, which were absent in uninfected controls.

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Environmental Sampling of Photosynthetic Microbes and Their Viruses: From Field to Lab

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