TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Purpose:</strong> To quantify virus-like particles in culture or environmental samples.</p>
<p><strong>Summary:</strong> Host cells and virus-like particles are enumerated from the same glutaraldehyde-fixed sample. For virus counts, an aliquot is diluted in 0.02-µm-filtered TE buffer prior to staining with SYBR Green I nucleic acid dye to improve staining efficiency and ensure an appropriately low event rate in the instrument. An unstained aliquot of sample is run for host cell counts.</p>

[BEFORE_START]
<p><strong>Equipment and Materials</strong>.</p>
<p><em> </em></p>
<p><em><u>Equipment</u></em><em>:</em></p>
<table>
<tbody>
<tr>
<td width="253">
<p>BD Influx Flow Cytometer</p>
</td>
</tr>
<tr>
<td width="253">
<p>Tube racks</p>
</td>
</tr>
<tr>
<td width="253">
<p>10, 20, 200, and 1000 µL pipettes</p>
</td>
</tr>
<tr>
<td width="253">
<p>Ice bucket</p>
</td>
</tr>
<tr>
<td width="253">
<p>Light-safe box for incubation</p>
</td>
</tr>
<tr>
<td width="253">
<p>Timer</p>
</td>
</tr>
<tr>
<td width="253">
<p>Vortexer</p>
</td>
</tr>
</tbody>
</table>
<p><em> </em></p>
<p><em><u>Materials</u></em></p>
<table style="width: 509px;">
<tbody>
<tr>
<td style="width: 499px;">
<p>10, 20, 200, and 1000 µL filter tips</p>
</td>
</tr>
<tr>
<td style="width: 499px;">
<p>High Intensity Green 0.5 µm fluorescent microspheres/beads (Polysciences #23516)</p>
</td>
</tr>
<tr>
<td style="width: 499px;">
<p>Plain YG 0.75 µm fluorescent microspheres/beads (Polysciences #17153)</p>
</td>
</tr>
<tr>
<td style="width: 499px;">
<p>1.7 and 2.0 mL microcentrifuge tubes</p>
</td>
</tr>
<tr>
<td style="width: 499px;">
<p>0.02-µm-pore-size sterile Anotop 25 Syringe Filter Plus (GE Healthcare #6809-4102)</p>
</td>
</tr>
<tr>
<td style="width: 499px;">
<p>0.2-µm-pore-size sterile Puradisc 25 PES Syringe Filter (GE Healthcare #6780-2502)</p>
</td>
</tr>
<tr>
<td style="width: 499px;">
<p>1X TE buffer  pH 8.0 (Fisher BioReagents #BP1338-1), 0.02-µm-filtered</p>
</td>
</tr>
<tr>
<td style="width: 499px;">
<p>1X PBS (BioPioneer #MB1001), 0.2-µm-filtered</p>
</td>
</tr>
<tr>
<td style="width: 499px;">
<p>5 mL round bottom polypropylene tubes</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong>Reagents and Solutions</strong>.</p>
<p> </p>
<table style="width: 640px;">
<tbody>
<tr style="height: 32px;">
<td style="width: 185px; height: 32px;">
<p><u>25% EM-grade glutaraldehyde</u>:</p>
</td>
<td style="width: 439px; height: 32px;">
<p>Prepare small aliquots (1 mL) to avoid cross-contamination and store at 4°C protected from light.</p>
</td>
</tr>
<tr style="height: 48px;">
<td style="width: 185px; height: 48px;">
<p><u>0.02-µm-filtered TE buffer</u>:</p>
</td>
<td style="width: 439px; height: 48px;">
<p>Dilute 0.5 mL 100X molecular grade Tris-EDTA (pH 8.0) in 49.5 mL MilliQ water. In a hood, filter through a 0.02-µm-pore-size sterile Anotop 25 syringe filter into sterile 15 mL tubes. Use within a few days of opening.</p>
</td>
</tr>
<tr style="height: 64px;">
<td style="width: 185px; height: 64px;">
<p><u>SYBR Green I working stock</u>:</p>
</td>
<td style="width: 439px; height: 64px;">
<p>Dilute concentrated 10,000X SYBR stock (Invitrogen # S7563 or S7567) to 100X in molecular grade DMSO (Sigma D8418) (i.e., 10 µl concentrated SYBR in 990 µL DMSO). Prepare small aliquots (30-50 µL) and discard after thawing. Staining efficiency decreases from freeze/thaw cycles (stable for 2-3 thaws). Briefly spin stock solution (20,000 x <em>g</em>) to reduce noise levels.</p>
</td>
</tr>
<tr style="height: 32px;">
<td style="width: 185px; height: 32px;">
<p><u>Green 0.5 µm beads</u>:</p>
</td>
<td style="width: 439px; height: 32px;">
<p>Prepare 2° stock by diluting 1 drop in 10 mL MilliQ water. Then prepare a 3° (working) stock by diluting 1 mL of 2° stock in 4 mL MilliQ.</p>
</td>
</tr>
<tr style="height: 48px;">
<td style="width: 185px; height: 48px;">
<p><u>YG 0.75 µm beads</u>:</p>
</td>
<td style="width: 439px; height: 48px;">
<p>Prepare 2° stock by diluting 1 drop in 10 mL MilliQ water. Then prepare a 3° stock by diluting 200 µL of 2° stock in 10 mL MilliQ. Finally, prepare a 4° (working) stock by diluting 1 mL of 3° stock in 9 mL MilliQ</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong> Dilution Table</strong>.</p>
<p> </p>
<p>Staining efficiency depends on having an appropriately diluted virus sample.</p>
<p> </p>
<table width="445">
<tbody>
<tr>
<td style="text-align: center;" width="75">
<p><strong>Dilution</strong></p>
</td>
<td style="text-align: center;" width="107">
<p><strong>Sub-dilutions</strong></p>
</td>
<td style="text-align: center;" width="98">
<p><strong>Sample Vol</strong></p>
</td>
<td style="text-align: center;" width="67">
<p><strong>TE Vol</strong></p>
</td>
<td style="text-align: center;" width="99">
<p><strong><em>Calc Check</em></strong></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="4" width="75">
<p>1:40,000</p>
</td>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:4</p>
</td>
<td style="text-align: center;" width="98">
<p>25</p>
</td>
<td style="text-align: center;" width="67">
<p>75</p>
</td>
<td style="text-align: center;" width="99">
<p><em>4</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="4" width="75">
<p>1:25,000</p>
</td>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:2.5</p>
</td>
<td style="text-align: center;" width="98">
<p>40</p>
</td>
<td style="text-align: center;" width="67">
<p>60</p>
</td>
<td style="text-align: center;" width="99">
<p><em>2.5</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="4" width="75">
<p>1:20,000</p>
</td>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:2</p>
</td>
<td style="text-align: center;" width="98">
<p>50</p>
</td>
<td style="text-align: center;" width="67">
<p>50</p>
</td>
<td style="text-align: center;" width="99">
<p><em>2</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="3" width="75">
<p>1:15,000</p>
</td>
<td style="text-align: center;" width="107">
<p>1:15</p>
</td>
<td style="text-align: center;" width="98">
<p>6</p>
</td>
<td style="text-align: center;" width="67">
<p>84</p>
</td>
<td style="text-align: center;" width="99">
<p><em>15</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="3" width="75">
<p>1:10,000</p>
</td>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="3" width="75">
<p>1:8,000</p>
</td>
<td style="text-align: center;" width="107">
<p>1:8</p>
</td>
<td style="text-align: center;" width="98">
<p>12.5</p>
</td>
<td style="text-align: center;" width="67">
<p>87.5</p>
</td>
<td style="text-align: center;" width="99">
<p><em>8</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="3" width="75">
<p>1:5,000</p>
</td>
<td style="text-align: center;" width="107">
<p>1:5</p>
</td>
<td style="text-align: center;" width="98">
<p>20</p>
</td>
<td style="text-align: center;" width="67">
<p>80</p>
</td>
<td style="text-align: center;" width="99">
<p><em>5</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="3" width="75">
<p>1:4,000</p>
</td>
<td style="text-align: center;" width="107">
<p>1:4</p>
</td>
<td style="text-align: center;" width="98">
<p>25</p>
</td>
<td style="text-align: center;" width="67">
<p>75</p>
</td>
<td style="text-align: center;" width="99">
<p><em>4</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="3" width="75">
<p>1:2,500</p>
</td>
<td style="text-align: center;" width="107">
<p>1:2.5</p>
</td>
<td style="text-align: center;" width="98">
<p>40</p>
</td>
<td style="text-align: center;" width="67">
<p>60</p>
</td>
<td style="text-align: center;" width="99">
<p><em>2.5</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="3" width="75">
<p>1:2,000</p>
</td>
<td style="text-align: center;" width="107">
<p>1:2</p>
</td>
<td style="text-align: center;" width="98">
<p>50</p>
</td>
<td style="text-align: center;" width="67">
<p>50</p>
</td>
<td style="text-align: center;" width="99">
<p><em>2</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="2" width="75">
<p>1:1,500</p>
</td>
<td style="text-align: center;" width="107">
<p>1:15</p>
</td>
<td style="text-align: center;" width="98">
<p>6</p>
</td>
<td style="text-align: center;" width="67">
<p>84</p>
</td>
<td style="text-align: center;" width="99">
<p><em>15</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="2" width="75">
<p>1:1,000</p>
</td>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="2" width="75">
<p>1:500</p>
</td>
<td style="text-align: center;" width="107">
<p>1:5</p>
</td>
<td style="text-align: center;" width="98">
<p>20</p>
</td>
<td style="text-align: center;" width="67">
<p>80</p>
</td>
<td style="text-align: center;" width="99">
<p><em>5</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="2" width="75">
<p>1:250</p>
</td>
<td style="text-align: center;" width="107">
<p>1:2.5</p>
</td>
<td style="text-align: center;" width="98">
<p>40</p>
</td>
<td style="text-align: center;" width="67">
<p>60</p>
</td>
<td style="text-align: center;" width="99">
<p><em>2.5</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:100</p>
</td>
<td style="text-align: center;" width="98">
<p>5</p>
</td>
<td style="text-align: center;" width="67">
<p>495</p>
</td>
<td style="text-align: center;" width="99">
<p><em>100</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" rowspan="2" width="75">
<p>1:100</p>
</td>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>10</p>
</td>
<td style="text-align: center;" width="67">
<p>90</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
<tr>
<td style="text-align: center;" width="107">
<p>1:10</p>
</td>
<td style="text-align: center;" width="98">
<p>50</p>
</td>
<td style="text-align: center;" width="67">
<p>450</p>
</td>
<td style="text-align: center;" width="99">
<p><em>10</em></p>
</td>
</tr>
</tbody>
</table>

[GUIDELINES]
<p><strong>Principle</strong></p>
<p> </p>
<p>Combining high-fluorescence-yield nucleic-acid-specific stains with flow cytometry has significantly improved the speed of analysis and accuracy of counting virus-like particles. Robust quantification using this method relies on reproducible staining efficiency as well as appropriate instrument parameters to ensure all events of interest are on scale. This protocol builds on the work of Brussaard (2004) <em>Appl Environ Microbiol</em>. (doi: 10.1128/AEM.70.3.1506-1513.2004), optimizing conditions specifically for enumeration of Prasinoviruses.</p>

[STEPS]
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