Team:Heidelberg/Notebook/Visualization/Methods

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[[Image:Small_chamber.jpg|left|thumb|300px|Small chamber 2x4 wells]][[Image:Large_chamber.jpg|left|thumb|300px|Large chamber]]
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===Small chamber===
===Small chamber===

Revision as of 21:01, 28 October 2008

Contents

Microscope

Was performed using LabtekTM chambered coverglass systems (Nunc). In most analysises mainly two versions were used.

Small chamber 2x4 wells
Large chamber


















Small chamber

beschriftung

chemotactical activity

beschriftung

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 or attractant line and the bacteria. !!!
    • 50 ml falcons
    • 25 ml media containig 0.2%-0.3% Agar for every petri-dish.
    • samples of the appropriate 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 attractant plugs1 or other non fluid attractants, go first through step 5 then back through 2,3,4 skip 5, further with 6 and so on...
  • Step 3: Take Take a 50 ml falcon pour 25 ml of your agar containing 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.: a casamino acid solution
  • 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 3-6 µl of your purrified bacteria at the premarked spots on the petri-dish sample.
  • Step 9: Put the whole plate into the incubator at ~30°C and take a look at your plates after 24, 48 and 72 hours. Put the plates into a plastic bag like the small ones for the bench trash to protect the plates from running dry to fast. Only move the plates if necessary.


1: Attractant plugs can be created by adding agar to the attractant solution.


Center attractant plug line
Spotted attractants
Center fluid attractant line
This picture is showing an alternative chamber
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