TITLE: No Title
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[DESCRIPTION]
<p><strong>Purpose:</strong> To quantify infectious viruses and evaluate the infectivity of a viral sample.</p>
<p><strong>Summary:</strong> 50 µL of serially-diluted (10<sup>-3</sup> to 10<sup>-10</sup>) virus sample is added to 150 µL of exponentially growing host cells in triplicate 96-well microplates and incubated at normal growth conditions for ~2 weeks. Cell lysis is assessed every few days qualitatively by visual inspection and quantitatively by measuring optical density on a microplate reader. The MPN of infective viruses in each concentrate is estimated from the proportion of virus-positive (i.e., lysed) wells using the <a href="http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich/MPN_ver4.xls" target="_blank">MPN_ver4.xls</a> Excel spreadsheet from Jarvis, B., Wilrich, C., and P.-T. Wilrich (2010), <em>Journal of Applied Microbiology</em> <strong>109</strong> (2010), 1660 – 1667. Percent infectivity is then calculated by comparing the MPN-estimated abundance of infective viruses to the abundance of virus-like particles (VLPs) determined by flow cytometry analysis.</p>

[BEFORE_START]
<p><strong>Equipment and Materials</strong>.</p>
<p><em> </em></p>
<p><em>Per virus sample tested:</em></p>
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<p>5 mL round bottom polypropylene tubes</p>
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<p>10</p>
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<p>Culture medium</p>
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<p>25 mL</p>
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<p>96-well microplates</p>
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<p>3</p>
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<p>Exponentially-growing host culture</p>
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<p>40 mL</p>
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<p>Virus sample</p>
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<p>0.2 mL (dilution) + 0.5 mL (FCM)</p>
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<p>Sterile 50 mL sample reservoirs</p>
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<p>2-3</p>
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<p>Cryovials</p>
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<p>2-3</p>
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<p> </p>
<p><em>Additional materials:</em></p>
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<p>1000 uL pipette + filter tips</p>
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<p>200 uL multichannel pipette + tips</p>
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<p>Tube rack</p>
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<p>Vortexer</p>
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<p>Optical microplate reader</p>
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<p>25% glutaraldehyde (EM grade)</p>
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<p> </p>

[GUIDELINES]
<p><strong>Principle </strong></p>
<p> </p>
<p>The abundance of infective viruses in a sample can be estimated using a serial dilution approach. In theory, a single lytic virus can lyse an entire population of sensitive host cells, given enough time. The MPN approach uses a series of log-based dilutions of a virus sample added to aliquots of an exponentially-growing host culture. The abundance of infectious viruses can be estimated if the range of dilutions used includes when the mean viruses per aliquot ≈ 1. An estimate of infective units is calculated with software using the number of lysed host aliquots at each dilution level. Replication (and increased sensitivity) is achieved by using 96-well microplates (8 replicate wells per plate x triplicate plates = 24 replicates per dilution). The estimated MPN is then compared to the total virus-like particle (VLP) count determined by flow cytometry to calculate infectivity (i.e., the proportion of infective viruses in the sample).</p>
<p> </p>
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<p><strong>Host Preparation</strong></p>
<p> </p>
<p>Initiate a host culture several days prior to assay setup to ensure exponential growth. Avoid transferring cells on the day-of or the day-before assay setup to minimize disturbance. Transfer/dilute host culture to pre-determined mid-exponential density daily (semi-continuous culture), including the day of assay setup.</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 ~100 µE m<sup>-2</sup> s<sup>-1</sup></p>
<p>Growth media: L1 with natural seawater base (67-135)</p>
<p>Exponential range: 7x10<sup>5</sup> – 2x10<sup>7</sup> cells mL<sup>-1</sup></p>
<p> </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>Initial density (mL<sup>-1</sup>)</strong></p>
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<p>5x10<sup>7</sup></p>
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<p><em>Skipped</em></p>
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<p>1.5x10<sup>7</sup></p>
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<p>9x10<sup>6</sup></p>
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<p>1.2x10<sup>7</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>0.55</p>
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<p>0.50</p>
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<p>0.84</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>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>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>(use for assay)</strong></p>
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<p><strong>Volume (mL)</strong></p>
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<p>15</p>
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<p>25</p>
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<p>44*</p>
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<p>104*</p>
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<p><em>*Volume varies depending on growth</em></p>

[STEPS]
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