Team:Heidelberg/Notebook/Visualization/Methods

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Revision as of 19:15, 28 October 2008

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. The concentration depends on several substance depending attributes.
- 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 In some cases
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/>

@@ Line 481: / Line 481: @@
 ** 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.
+** 25 ml media containig 0.2%-0.3% Agar for every petri-dish.
 ** samples of the apropriate attractants. The concentration depends on several substance depending attributes.
 ** Other substances like antibiotics (Amp, Kan, Cm) or inducer (IPTG, Arabinose)