Team:TU Darmstadt/protocols/Cell counting/plating

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<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font>
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<h2><font size="6" color="#F0F8FF" face="Arial regular"> Cell counting/plating </font></h2>
<h2><font size="6" color="#F0F8FF" face="Arial regular"> Cell counting/plating </font></h2>
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<B> Materials<br></B></font></p>
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<B> Materials<br></B>
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LB agar plate<br>
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LB media<br>
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tubes<br>
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<B> Procedure<br></B>
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<li>Fill each tube in the dilution with 90 &mu;l of LB</li>
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<li class=list1>- LB agar plate</li>
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<li>Add 10 &mu;l of the sample to the first tube and mix</li>
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<li class=list1>- <a href="https://2013.igem.org/Team:TU_Darmstadt/materials/Media"> LB media</a></li>
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<li>From the first tube, remove 10 &mu;l and mix into second tube</li>
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<li class=list1>- Tubes</li>
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<li>Repeat for the number of dilutions you wish to do (8 should be more than enough)<sup>[2]</sup></li>
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<li>Take 10 &mu;l from each dilution and spot it on to the agar plate</li>
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<li>Allow droplet to dry and incubate</li>
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The first dilutions will contain a thick lawn of cells and the last dilutions will contain no cells. There should be one drop which contains countable single colonies. From this, you can calculate the number of cells in the original sample. For example, if there 4 colonies on dilution 5, there are 4E4 cells/&mu;l.<br>
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<B> Procedure<br></B></font></p>
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<B>References<br>
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http://openwetware.org/wiki/Cell_counting/plating
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<li>Fill each tube in the dilution with 90 &mu;l of LB.</li>
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<li>Add 10 &mu;l of the sample to the first tube and mix.</li>
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<li>From the first tube, remove 10 &mu;l and mix into second tube.</li>
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<li>Repeat for the number of dilutions you wish to do (8 should be more than enough) <sup><span style="color:blue">[1]</span></sup>.</li>
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<li>Take 10 &mu;l from each dilution and spot it on to the agar plate.</li>
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<li>Allow droplet to dry and incubate.</li>
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</ol>
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</div></font></p><br>
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<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular">
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The first dilutions will contain a thick lawn of cells and the last dilutions will contain no cells. There should be one drop which contains countable single colonies. From this, you can calculate the number of cells in the original sample. For example, if there 4 colonies on dilution 5, there are 4E4 cells/&mu;l.</font></p>
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<br><h2><font size="6" color="#F0F8FF" face="Arial regular">References</font></h2>
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<font size="3" color="#F0F8FF" face="Arial regular">
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<ol>
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<li style="margin-left:15px; margin-right:50px; text-align:justify">http://openwetware.org/wiki/Bacterial_cell_culture</li>
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Latest revision as of 00:30, 5 October 2013







Lab book | Materials | Protocols

Cell counting/plating

Materials


Procedure

  1. Fill each tube in the dilution with 90 μl of LB.
  2. Add 10 μl of the sample to the first tube and mix.
  3. From the first tube, remove 10 μl and mix into second tube.
  4. Repeat for the number of dilutions you wish to do (8 should be more than enough) [1].
  5. Take 10 μl from each dilution and spot it on to the agar plate.
  6. Allow droplet to dry and incubate.


The first dilutions will contain a thick lawn of cells and the last dilutions will contain no cells. There should be one drop which contains countable single colonies. From this, you can calculate the number of cells in the original sample. For example, if there 4 colonies on dilution 5, there are 4E4 cells/μl.


References

  1. http://openwetware.org/wiki/Bacterial_cell_culture

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