Chlorella Virus Plaque Assay

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<div> 
 <h1>Chlorella Virus Plaque Assay</h1> Imported from 
 <a href="http://ncv.unl.edu/vanettenlab/Plaque%20assay.pdf" target="_blank" rel="noopener">Protocols.io</a> 
 <h2>Metadata</h2> 
 <strong>Original creator:</strong> 
 <a href="http://ncv.unl.edu/vanettenlab/Plaque%20assay.pdf" target="_blank" rel="noopener">Steven W. Wilhelm</a> 
 <br> 
 <strong>Published on:</strong> Wed Mar 29 14:50:25 UTC 2017 
 <br> 
 <strong>Original authors:</strong> Dr. Steven Wilhelm , Samantha Coy 
 <br> 
 <strong>Version:</strong> 1 
 <h2>Steps</h2> 
 <h3 data-step-id="410104"><a id="step-410104"></a>Step 1</h3> 
 <h4>MBBM Soft Agar</h4> 
 <br> 
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  <div class="text-block">
    Melt MBBM soft agar and dispense&nbsp;in 3.0 mL aliquots and hold at 45ºC - 50ºC in a water bath 
  </div> 
 </div> 
 <br> 
 <p>&nbsp;</p> 
 <h3 data-step-id="410105"><a id="step-410105"></a>Step 2</h3> 
 <h4>Concentrate Chlorella</h4> 
 <br> 
 <div class="text-blocks"> 
  <div class="text-block">
    Concentrate Chlorella to 4.0*10 
   <span style="vertical-align: super;">8&nbsp;</span>cells/mL at 5,000 rpm for 5 min at 4ºC and resuspend with fresh MBBM 
  </div> 
  <div class="text-block">
    *Concentrate so that 0.3 mL can be used per plate 
  </div> 
 </div> 
 <br> 
 <p>&nbsp;</p> 
 <h3 data-step-id="410106"><a id="step-410106"></a>Step 3</h3> 
 <h4>Dilute Chlorella Virus</h4> 
 <br> 
 <div class="text-blocks"> 
  <div class="text-block">
    Dilute virus with 50 mM Tris-HCl, pH 7.8 in 1/10 serial dilutions 
  </div> 
  <div class="text-block">
    *Fresh lysate contains approximately 10 
   <span style="vertical-align: super;">6</span> -10 
   <span style="vertical-align: super;">7</span> PFU/mL (check the titer prior to plaque assay) 
  </div> 
  <div class="text-block">
    *Dilute sample to have 20-200 plaque forming units (PFU) per plate 
  </div> 
 </div> 
 <br> 
 <p>&nbsp;</p> 
 <h3 data-step-id="410107"><a id="step-410107"></a>Step 4</h3> 
 <h4>Titering</h4> 
 <br> 
 <div class="text-blocks"> 
  <div class="text-block">
    To each 3.0 mL soft agar aliquot, add 0.1 mL Chlorella virus and 0.3 mL Chlorella 
  </div> 
  <div class="text-block">
    *Mix by rolling between palms and pour onto MBBM plate 
  </div> 
  <div class="text-block">
    *Tilt the plate gently to allow the entire surface to be covered 
  </div> 
  <div class="text-block">
    *Allow plate to solidify 
  </div> 
 </div> 
 <br> 
 <p>&nbsp;</p> 
 <h3 data-step-id="410108"><a id="step-410108"></a>Step 5</h3> 
 <h4>Incubation</h4> 
 <br> 
 <div class="text-blocks"> 
  <div class="text-block">
    Incubate at 25ºC in continuous light for 6 days or until plaques are visible to count 
  </div> 
  <div class="text-block">
    *Use a sharpie to count plaques once there is a visible contrast 
  </div> 
 </div> 
 <br> 
 <p>&nbsp;</p> 
</div>

Last modified: Thu Mar 26 15:20:30 UTC 2020

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Chlorella Virus Plaque Assay

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