"

 

From 2008.igem.org

Contents 1 Microscope 1.1 Small chamber 1.1.1 chemotactical activity 1.2 Large chamber 1.3 computer-assisted analysis 2 Swarm Assays

Microscope

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

Small chamber

asdasdasdasdasdasdasdasdasd

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 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.

• Recent changes
• What links here
• Related changes
• Special pages
• My preferences

• Printable version
• Permanent link
• Privacy policy
• Disclaimers