I am attempting to grow E.coli biofilms using 96, and 24 well flat bottom plates using the attached M9 media recipe. Using 400 uL of media in 24 well plates, and 150 uL of media in 96 well plates. Plates are incubated at 37°C for approximately 24 hours. Bacteria growth is visible but usually only at the top of media, but after washing with PBS plate and adding XTT (incubating for 2hrs) absorptions are very low. Am I washing the biofilms out? Not incubating long enough? To much/little media?
I am going to try incubating plates at 30°C per advice given, but how much media and for how long?
Any help or suggestions would be very much appreciated.
Hello,
What kind of plate do you use ? Aren't they treated againt coating ?
Which strain of E coli do you use ? Biofilm formation change a lot between two different strains, you may just have a low biofilm producer strain (bad luck).
If you change your incubation temperature you will need to incubate for a longer time (48 hours at 30°C, and 4-5 days at RT).
You can try to change the media (adding Casaminoacids or yeast extract if it's possible) or incubating for a longer time (48h for example).
About the plates, there is a huge difference between 28, 48 and 96 well plates for biofilm formation. I recommend you to use always the same plate "size" and do not try to compare results obtained with different plates "size".
I don't know if you are using shaking incubation but it can reduce biofilm formation.
Some years ago I did some biofilm formation using TTC (XTT), if you want a protocol you can find it there : Article Critical Assessment of Methods to Quantify Biofilm Growth an...
Have a nice day
Quentin
Quentin Vallé
No I believe that the plates we're using are un treated. I'll try and find out more about the plates.
Key points for biofilm cultivation are:
1. Biofilm innoculum is usually an exhausted stationary phase culture which has been shaken vigourously overnight. This is diluted 1/50 or 1/100 in half stregth media to seed the actual biofilm. Volume in 96 well plate usually 150-200ul.
2. Use Hi-bind plates or ELISA/cell culture treated plates from COSTAR because the plastic is more compatable with binding organic molecules.
3. Cultures are usually incubated static on a level surface.
4. Like the commentator above I do several pilot experiments to confirm my organism is a robust producer of biofilm. If not i will vary the incubation temperature.
5. after biofilm growth I crefully aspirate the growth media.
6. I leave the plate to air dry for 10 minutes and then rinse very gently with PBS. this depends on the robustness of the biofilm.
7. I stain the biofilm with crystal violet , rinse with PBS and then transfer the stain with 30% acetic acid to another plate.
8. Make sure you have control media and control blank wells.
I suspect you might be dealing with biofilms which form on top of a surface. I have just started cultivating these and it requires a slightly different technique. Especially when aspirating, come from the bottom corner of the well with a very fine pippette tip. Leave biofilms to dry for longer.
I recommend starting off with a 96 well plate, test optimal strains and conditions and then you can move to 24 or 12 well plates. I have found you can make the area to large for certain biofilms to fill .
hope that helps .
i published some literature on biofilm testing a few years ago .
Article Microbial biofilms: Biosurfactants as antibiofilm agents
Quentin Vallé Gerry A Quinn ,
Thank you all so much for your help. I will try vary the temperature and move forward from there. Thanks again, I really appreciate the suggestions and help!
We just published a method for kinetic measurement of bacterial attachment in 96 well plates that doesn't involve staining or whasing. This may help with a weak biofilm - link below
https://link.springer.com/article/10.1007/s00248-018-1254-5
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