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Team:Heidelberg/Notebook/Visualization/Methods
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PROTOCOL FOR SWARM-PLATE CHEMOTAXIS AGAINST A POSSIBLE ATTRACTANT:<br> | PROTOCOL FOR SWARM-PLATE CHEMOTAXIS AGAINST A POSSIBLE ATTRACTANT:<br> | ||
| - | + | This protocol was created using petri-dishes. But every other chamber could be used as well. | |
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*Step 1: Make sure you have everything ready to get started. You need: | *Step 1: Make sure you have everything ready to get started. You need: | ||
** Petri-dishes. '''!!!''' ''sign your petri-dishes before You pour in the media. Also mark the spots where you want to set the attractant and the bacteria.'' '''!!!''' | ** Petri-dishes. '''!!!''' ''sign your petri-dishes before You pour in the media. Also mark the spots where you want to set the attractant and the bacteria.'' '''!!!''' | ||
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EXAMPLES:<br> | EXAMPLES:<br> | ||
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Attractants: | Attractants: | ||
<references/> | <references/> | ||
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Revision as of 19:08, 28 October 2008
Contents |
Microscope
Small chamber
chemotactical activity
Large chamber
computer-assisted analysis
Swarm Assays
PROTOCOL FOR SWARM-PLATE CHEMOTAXIS AGAINST A POSSIBLE ATTRACTANT:
This protocol was created using petri-dishes. But every other chamber could be used as well.
- Step 1: Make sure you have everything ready to get started. You need:
- Petri-dishes. !!! sign your petri-dishes before You pour in the media. Also mark the spots where you want to set the attractant and the bacteria. !!!
- 50 ml falcons
- 25 ml media containig 0.2%-0.3% Agar for every petri-dish.
- samples of the apropriate attractants.
- Other substances like antibiotics (Amp, Kan, Cm) or inducer (IPTG, Arabinose)
- Step 2: Your agar-media solution should be clear and the agar completely solved. If you want to use casamino acid plugs as attractant go first through step 5 then back through 2,3,4 skip 5,6 and so on...
- Step 3: Take Take a 50 ml falcon pour 25 ml of your agar-media in it and add antibiotics or inducing substances. If no extra substances are needed just skip this step.
- Step 4: Let your media polimerize. At least let it rest for 15/20 min.
- Step 5: Then add the attractant. e.g.: casamino acid plug<ref>casamino acid plug
I used a solution containing 10% casamino acids and 2% agar called CA2A
Take a scalpel and cut off the upper part of a yellow pipette peak and put it upside down on your petri dish, there where you want the attractant plug to be
Heat the CA2A solution until it is fluid and the agar completely solved.
Put 100 µl of the CA2A solution inside the tube-top.
Let it rest for 10 minutes, then "gently" push the plug out of the tube-top, therefor you may use a blue pipette peak.
There should still be a few samples of CA2A in the second -20 fridge, first shelf, blue rag, labeled GX. To get it fluid put it into the heating cube and after that a few times into the microwave, but just for 3-5 seconds, NOT at all longer!!! Kepp the tube away from you and open it gently after everytime you put it into the microwave.</ref> - Step 6:
- Put the whole plate into a 4°C fridge for 12-16 hours to allow the attractant to build up a gradient.
- Make an overnight culture of the strains you want to test, use TB media and add inducing substances if something has to be induced.
Next day:
- Step 7: Take your overnight culture. It should be at an OD of 0.4-0.6. Then take 1 ml of the bacteria suspension and centrifuge it for 10 minutes at 4,000 rpm. Resolve the pellet at 100 µl of the media you are using in the petri-dish.
- Step 8: Place 4 µl of your purrified bacteria at the premarked spot on the petri-dish sample.
- Step 9: Put the whole plate into the incubator at ~30°C for 24 hours.
EXAMPLES:
Attractants:
<references/>
