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Team:ZJU-China/Notebook/Biofilm
From 2011.igem.org
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Early July:
Pre-experiments with biofilm formation with circular and non circular silicone tube, 24 well plate on different support including glass, paper, plastic film, rubber. The final material are used based on the easiness biofilm form on them and on the easiness to observe under microscope.
28th July:
13.30: DH5ɑ 11p 5mlX4
23.00: silicone tube set at 37℃
29th July:
13.00: LB culture 50ml with circular culture medium. LB culture 50ml with noncircular culture medium.
31st July:
13.00: -80℃ storing silicone tube. A thick white and loosely bond substance is seen on the inner wall of the 5mm silicone tube, and on the inner wall of the 1mm silicone tube a flatter and smoother white substance. Especially obvious where the tube turns. Possibly because the speed of culture flow is slower.
1st August
Freeze slicing of about 50μm. Observe under natural light microscope and can see red florescence. The thickness is about 130μm
4th August
Repeat the last experiment with silicone tube. 6rpm/s results in a flowing speed of 68ml/day
6th August
Similar to the last time. Did not use freeze slicing.
15th August
Cultured E.coli in 5ml LB for 12h.
16th August
Substituting a part of silicone tube with glass tube in silicone tube biofilm formation sets. Culture in 37℃
17th August
Add 50ml LB and culture with circular culture.
18th August
12.00 found one free-flow pump stopped. Cooled for 15 minutes and turned on again. Could be over heated. Can see some bacteria on the bottom of the vessel and tube. When the pump started again, the bacteria was washed away.
19th August
Can observe obvious white biofilm where the silicone tube joins the tube but cannot see red florescence, suspect contamination. No biofilm is observed on the glass tube under microscope.
21st, August
Biofilm formation with large test tubes. Inoculate with 1% e.coli. Rubber tubes are attached to air
pump with filter between air pump and the tube to prevent entrance of germs. Two sets use MSM
+glucose medium and two sets use LB.
Retry with glass tube biofilm formation set.
24th, August
Terminate biofilm formation, the glass slide left in the drawer to dry. Can see obvious rod like
structure under 400 magnification. (spheres in MSM+glucose culture) No red florescence is seen.
Most of the surface is covered by single layer cells but some part of it has thick bump like
structure.
No biofilm observed on the glass tube.
3rd Sep
Directly use DH5α fdhF+RFP colony to inoculate silicone tube set with 5ml LB. rest for 12h for biofilm attachment.
4th Sep
Add 50mlLB+Amp and start circulation.
6th Sep
Biofilm with direct inoculation is optimum. Expressed red florescence but only one small fraction of the biofilm remained after cutting off the part of the tube.
12th Sep
4 silicone tube sets with biofilm attachement time of 3h, 5h, 7.5h and 10h; 4 bubbling sets.
15th Sep
The silicone tube set with 3h of attachment achieves optimum biofilm formation. Bubbling sets glass slides are froze in -80℃ for later observation with laser confocal. Biofilm formed on glass slide is about 10μm thick.
16th Sep
New sets with silicone tube, a mixture of RFP, CFP and YFP expressing e.coli. Attachment 3h.
17th Sep
Glass slide with bubbling set with the same mixture of e.coli.
Place two filter film on solid culture medium and placed the mixture of e.coli on the filter to form
gas-solid interface biofilm.
20th Sep
Observation of glass slide and gas-solid biofilm under laser confocal. Glass slide are observed
directly and gas-solid biofilm is first pressed by a covering slide so that it would stick to it and
peeled off from the filter for observation.
Biofilm on glass slide is about 8.6μm thick. Biofilm formed on gas-solid interface is too
concentrated in bacteria and culture medium that the florescence of too strong to see any clear
structure.
