TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Purpose:</strong> To reproducibly generate fresh preparations of virus for independent experiments.</p>
<p><strong>Summary:</strong> Fresh virus sample for an experiment is generated by a primary infection of exponentially growing host cells from a master stock of virus. The infected host is allowed to lyse until the culture is cleared. The lysate is filtered to remove any large cellular debris, then the viral-size fraction is concentrated from the filtered lysate and washed with buffer using a centrifugal concentrator.  The viral concentrate is stored at 4°C protected from light and should be used for an experiment within 1-2 days. On the day of (or the day before) the experiment, an MPN assay should be set up to assess infectivity of the fresh viral concentrate.</p>

[BEFORE_START]
<p><strong>Equipment and Materials</strong>.</p>
<p> </p>
<p><em><u>Equipment</u></em><em>:</em></p>
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<p>Tube rack</p>
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<p>200 and 1000 µL pipettes</p>
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<p>Pipettor for serological pipettes</p>
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<p>Refrigerated benchtop centrifuge capable of 1,000 x g</p>
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<p>Swinging bucket rotor that can accommodate 50 ml conical bottom tubes</p>
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<p>Vortexer</p>
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<p> </p>
<p><em><u>Materials</u></em><em>:</em></p>
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<p>Host culture</p>
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<p>Virus master stock</p>
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<p>Culture medium</p>
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<p>200 and 1000 uL filter tips</p>
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<p>Serological pipettes</p>
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<p>0.45-µm-pore-size PES membrane Nalgene Rapid-Flow Sterile Disposable Filter Unit(s) (Thermo Scientific #166-0045)</p>
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<p>100 kDa MWCO PES membrane VivaSpin20 ultrafiltration unit (Sartorius #VS2041)</p>
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<p>15 or 50 mL conical tubes, sterile</p>
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<p>Parafilm</p>
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<p>0.02-µm-pore-size sterile Anotop 25 Syringe Filter Plus (GE Healthcare #6809-4102)</p>
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<p>1X TE buffer  pH 8.0 (Fisher BioReagents #BP1338-1), 0.02-µm-filtered (see below)</p>
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<p> </p>
<p> </p>
<p><strong> Preparation of TE buffer (1X, pH 8.0)</strong>.</p>
<p> </p>
<p>Dilute 0.5 mL 100X molecular grade Tris-EDTA (Fisher BioReagents #BP1338-1) in 49.5 mL MilliQ water. In hood, filter through 0.02-µm-pore-size sterile Anotop 25 Syringe Filter Plus (GE Healthcare #6809-4102) into sterile 15 mL tubes. Use within a few days of opening.</p>

[GUIDELINES]
<p><strong>Principle</strong></p>
<p> </p>
<p>Several external physical and chemical factors (e.g., temperature, salinity, acidity, light, and adsorption) influence the morphology, persistence, and function of marine viruses. Because viruses depend on their hosts for replication, biological parameters such as host physiology and growth rate, as well as viral and host abundances (i.e., associated encounter rate), likewise impact the properties of a particular viral population (e.g., infectivity, abundance). Standardized and characterized host growth prior to infection combined with consistent infection parameters (e.g., host density, viral volume added, time allowed for lysis, incubation conditions, etc.) should minimize variation among viral preparations and improve reproducibility of viral properties among experiments. Furthermore, using the same master stock of virus for each propagation instead of a serially-propagated virus stock should reduce the probability of introducing genetic mutations.</p>
<p> </p>
<p> </p>
<p><strong>Host Preparation</strong></p>
<p> </p>
<p>Initiate a host culture for the primary infection to allow for at least 5 generations of characterized exponential growth (10 generations is the gold standard). Avoid transferring cells on the day-of or the day-before the primary infection to minimize disturbance. Monitor growth and transfer/dilute host culture to pre-determined mid-exponential density daily (i.e., semi-continuous culture), including the day of the primary infection. If the host culture will be split to propagate multiple viruses simultaneously, make sure this is done at least 2 days prior to addition of virus. After final dilution of the host culture to pre-determined mid-exponential density, initiate primary infection as described below.</p>
<p> </p>
<p><u>Example <em>Ostreococcus lucimarinus</em> (CCMP2972A) host preparation:</u></p>
<p>Growth conditions: 18°C, 14:10 hour light:dark cycle, light irradiance of ~100 µE m<sup>-2</sup> s<sup>-1</sup></p>
<p>Growth media: L1 with natural seawater base</p>
<p>Exponential range: 7x10<sup>5</sup> – 2x10<sup>7</sup> cells mL<sup>-1</sup></p>
<p> </p>
<p>For 2 viruses, intermediate volume (from Oct 2015): ~6 generations of characterized growth</p>
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<p><strong>Day</strong></p>
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<p><strong>1</strong></p>
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<p><strong>2</strong></p>
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<p><strong>3</strong></p>
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<p><strong>4</strong></p>
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<p><strong>5</strong></p>
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<p><strong>6</strong></p>
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<p><strong>7</strong></p>
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<p><strong>Initial density (mL<sup>-1</sup>)</strong></p>
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<p>3.5x10<sup>7</sup></p>
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<td width="54">
<p>9x10<sup>6</sup></p>
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<td rowspan="5" width="48">
<p>Skip</p>
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<td width="72">
<p>1.7x10<sup>7</sup></p>
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<td width="72">
<p>7x10<sup>6</sup></p>
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<p>9.4x10<sup>6</sup></p>
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<p>9.4x10<sup>6</sup></p>
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<p><strong>Growth Rate (d<sup>-1</sup>)</strong></p>
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<p>NA</p>
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<p>0.68</p>
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<p>0.69</p>
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<p>0.53</p>
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<p>0.62</p>
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<p>0.68</p>
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<p><strong>Dilution/Transfer</strong></p>
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<p>Transfer</p>
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<p>Dilute</p>
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<p>Transfer</p>
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<p>Transfer <em>(split)</em></p>
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<p>Dilution</p>
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<p>Dilution</p>
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<p><strong>Final density (mL<sup>-1</sup>)</strong></p>
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<p>4x10<sup>6</sup></p>
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<p>4x10<sup>6</sup></p>
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<p>5x10<sup>6</sup></p>
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<p>5x10<sup>6</sup></p>
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<p>5x10<sup>6</sup></p>
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<p>5x10<sup>6</sup> <strong>(infect!)</strong></p>
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<p><strong>Volume (mL)</strong></p>
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<p>60</p>
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<p>137*</p>
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<p>200</p>
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<p>140 <em>x2</em></p>
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<p>260* <em>x2</em></p>
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<p>490* <em>x2</em></p>
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<p> </p>
<p>For 2 viruses, large volume (from Apr 2016): ~5 generations of characterized growth</p>
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<p><strong>Day</strong></p>
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<p><strong>1</strong></p>
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<p><strong>2</strong></p>
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<p><strong>3</strong></p>
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<p><strong>4</strong></p>
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<p><strong>5</strong></p>
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<p><strong>6</strong></p>
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<p><strong>7</strong></p>
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<p><strong>Initial density (mL<sup>-1</sup>)</strong></p>
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<p>2.2x10<sup>7</sup></p>
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<p>Skip</p>
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<p>Skip</p>
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<p>1.8x10<sup>7</sup></p>
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<p>8.7x10<sup>6</sup></p>
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<p>8x10<sup>6</sup></p>
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<p>8.7x10<sup>6</sup></p>
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<p><strong>Growth Rate (d<sup>-1</sup>)</strong></p>
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<p>NA</p>
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<p>0.760</p>
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<p>0.516</p>
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<p>0.481</p>
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<p>0.487</p>
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<p><strong>Dilution/Transfer</strong></p>
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<p>Transfer</p>
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<p>Transfer</p>
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<p>Transfer <em>(split)</em></p>
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<p>Dilution</p>
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<p>Dilution</p>
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<p><strong>Final density (mL<sup>-1</sup>)</strong></p>
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<p>4x10<sup>6</sup></p>
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<p>5x10<sup>6</sup></p>
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<td width="72">
<p>5x10<sup>6</sup></p>
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<td width="66">
<p>5x10<sup>6</sup></p>
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<p>5x10<sup>6</sup> <strong>(infect!)</strong></p>
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<p><strong>Volume (mL)</strong></p>
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<p>120</p>
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<p>400</p>
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<p>330 <em>x2</em></p>
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<p>534* <em>x2</em></p>
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<p>840* <em>x2</em></p>
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<p><em>*Volume varies depending on growth</em></p>

[STEPS]
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