Team:Groningen/protocols/Motility assay

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<h2>Motility assay</h2>
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<p>A motility assay is done for <i>Bacillus subtilus</i> of different knockout strains as wel as a wild type strain.  
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<h1>Motility assay</h1>
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<p>This is to determine how different the knockouts move compared to the wild type <i>B. subtilus</i>.
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Add 13 ml of the motility agar (0.4%) in every petri dish.
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<p>The results will be used to determine if the temperature controlled strain works as desired, moving more as a CheY knockout in warm environments and more as a wild type in cold environments.
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<br>Inoculate the plates with the strain of interest (10 &micro;l of overnight culture with an OD<sub>600</sub> of 0.4).
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<p>The design of the motility assay is a simple one. LB agar Plates are made with the normal amount of LB-broth as nutrients but with a reduced amount of agar.  
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<br>The spot is dried for half an hour, after which the plates are inoculated at different temperatures (25&deg;C and 37&deg;C) for 16 hours.
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<p>Different concentrations are used to find out the optimal amount of agar to show the motility.
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<br>After 16 hours of growth, the diameter of the colony is measured.  
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Low concentrations are needed to allow movement through the medium but too low and the observed movement can be caused by dispersal and turbulence during movement.
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The concentrations that are used are, 0.4% agar and 0.7% agar. The plates contained 13 ml agar.
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<p>
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<p>The plates are inoculated in the center with the strain that is to be tested. This is done with 10 ul of liquid culture at an OD of 0.4. The inoculation was done by sticking the pipet in to the agar, injecting the sample in to it.
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<b>Motility Agar 0.4% (100 ml) </b>
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<p>For each strain 9 plates of each agar concentration is made. Three of the plates are placed in a 37 C stove and three placed in a 30 C stove and three placed in a 27 C stove to grow.
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</p>
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<p>The colonys are left to grow for 16 hours. Then how the colonies spread on the plates are observed every hour. The bacteria that are more motile should spread out over the agar creating a clouwdy look while the non- motile bacteria should stay in the center, close together.  
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<p>How quickly the bacteria spread from the center to the edge can be used as an indicatore of how fast they move.
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<table id="normal" width="60%">
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  <tr>
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    <th>compound</th>
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    <th>amount</th>
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  </tr>
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  <tr>
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    <td>LB broth </td>
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    <td>2 g</td>
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  </tr>
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    <td>agar</td>
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    <td>0.4 g</td>
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  </tr>
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</table>
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Latest revision as of 00:22, 5 October 2013

Motility assay

Add 13 ml of the motility agar (0.4%) in every petri dish.
Inoculate the plates with the strain of interest (10 µl of overnight culture with an OD600 of 0.4).
The spot is dried for half an hour, after which the plates are inoculated at different temperatures (25°C and 37°C) for 16 hours.
After 16 hours of growth, the diameter of the colony is measured.

Motility Agar 0.4% (100 ml)

compound amount
LB broth 2 g
agar 0.4 g