In addition to biofilm inhibition in static culture, pregrown could be disrupted and cleared by switching to rich - media in flow chamber experiments. .......time, plates were rinsed to remove spent media, compounds, and planktonic cells, leaving only biofilm bacteria. For more information consult https://www.sciencedirect.com

in my expertise the best way is perform liquid aspiration using vacuum with a tube carryng a tip (eg a gilson 200ul yellow tip) at the end. you have to remove liquid 1 by 1 by crawling on the side of well from the top to the bottom.

however in my experience to obtain good p.aeruginosa film in multiwell you need to optimize plastic..medium and incubation time.i briefly worked on it more than 10 years ago but if i remenber well differently to s.aures that like rich media for aureuginosa i obtained better result with chemically defined medium. So is also possible that of your biofilm is so fragile is not a Real biofilm and you need to futher optimize it growth.

good luck

Manuele

And If you to use a fluid as substrate? I have been making biofilms of kefir and i use destiled water for forming the biofilms. Why you don't use water and sugar as substrate? In this form Will be more easy to remove the biofilm of the media

Thank you for your responses. I am moving to a defined MOPS media for the culture, so am hoping that this alleviates some of the issues. I will attempt a microlitre by microlitre removal approach and see if it helps.

Dear researcher,

I lived your case and I felt bad until I saw JOVE videos like :

Article A Platform of Anti-biofilm Assays Suited to the Exploration ...

I hope it will serve you

Dear Jonhatan

i do not know if it could be to usefull to you but this was the protocooll the i used:

- Day 1 afternoon: - P.aeruginosa PA01 inoculated in 5 ml of Tryptic Soy Broth (TSB) form glycerol stock and growth 16-18 h at 37°C- 150-180 rpm. -

Day 2 morning: - Cells were centrifuged (4000 rpm, 10’), washed 2 times with 5ml PBS1X and re-suspended with a volume of Low Glucose Media to obtain a OD(600nm) of about 0,05 (about)1,0x107 CFU/ml. - 200µl di this bacterial suspension are placed in each well of a GREINER - Cellstar Microplate 96Well White cod. 655083), plates were closed with a film and incubated at 37°C and basculating movement. -

Day 3 morning: - After 24h the exausted media is removed and replaced with other 200ul of fresh media and incubated for other 24h a 37°C (biofilm maturation step). -

Day 4 morning: - Media is discharged. Biofilm is washed 3 times with 100ul for well of sterile PBS and biofilm is ready for analysis ( eg CV /XTT)

where low glucose media is: 1,5% tryptone, 0,1% glucose dissolved in PBS ph=7,2

You could insert a sterile thin tube before inoculation, then remove the culture medium.

For P. aeruginosa biofilm in our lab we used M9 minimal medium. "Biofilm" grown in standard rich broth are just agglutinated cells, so these are easy to remove.

Hi Jonhatan,

In my experience, the robustness of biofilm attachment to the surface is higly dependent on plastic composition. I tried several brands and get the best results for PA01 and PAK using cellstar plates, cat. number 655 180. To remove the liquid I used a strerile Pasteur pipete.

Best,

Jessica

Use minimal media (MOPS is a good one). Otherwise, the polysaccharide amount is too much and the media is just slime and is very hard to aspirate out without damaging the biofilm structure. There is still a lot of biofilm formed not just aggregates even in rich media however, it is hard to quantify in these rich media because it is easy to damage the biofilm structure.

When I really have to use rich media for these assays. I use a multi-channel pipet and carefully aspirate the "slime" out (takes some time if you have a ton of plates). If you put the pipet tips all the way to the bottom of the well and do it slowly there is minimal damage to the biofilm structure. PAO1 biofilms forms at air/liquid interface usually; if done slowly you can pull out the media with limited damage. This doesn't work well with bacteria that form biofilms at the bottom of the wells because the tips damage the bottom biofilms.

Good luck!

This is exactly the course of action I followed today. Did you end up coming up with a good protocol? I would be really interested to know.

Hannah Dziura Not yet, as I have been working on other things. I have used MOPS defined medium, but biofilm formation is slow/minimal. I moved to larger volumes in 24-well trays, but think that the key could be incubation on a rocking device. More work is required, but I will report back when I have a working protocol.

Jonathan Vernon - you are interested in the biofilm that forms at the air/liquid interface and not the (less visible) biofilm at the liquid/surface interface, yes?

Another good way might be a microscope slide in a 50 mL centrifuge tube. If you don't mind going to the large volumes required for these (usually I use 25 mL). The other good thing about these is the ability to transfer the slide to a new tube with fresh media. This allows growth over multiple days/week without completely starving the biofilm. I do grow these with rocking (usually a shaker set to 100 rpm).

Something like in this link: https://www.vanderbilt.edu/vicb/images/discovery/dissecting_structure_biofilms_fig2.jpg

I actually have 50 of these tubes going right now with PAO1 and it is giving some really robust biofilms on the slides.

You can also try this method with the 24 well plate and use small plastic/glass cover slips. It seems to work but it depends on what you want to do with the biofilm or how high throughput you need it to be.

Donald Hall - that looks amazing!!! Do you know of any publications that have used this technique? I’d love to see what kind of data they obtained, and the protocol they used! I’d be keen to try it today even, since I have some P. aeruginosa culture ready to go :P

Hi Hannah,

Here a slightly adapted method from one of my mentor's papers(link here too). He was using E. coli but the protocol is very similar for P. aeruginosa. The only thing that I recommend is when you wash the cells be very careful because some of the biofilms will slough off (I'm working on a few ways to prevent this now by changing the washing solution. I think the saline and even just water is too polar and it's making the biofilm jump off if you submerge it quickly).

He was imaging them right away in his protocol because he was looking at plasmid transfer. I have stained with CV, or used 3 % glutaraldehyde to fix the biofilm for SEM imaging both work great.

Article Increased Transfer of a Multidrug Resistance Plasmid in Esch...

Batch culture biofilm reactor (see Fig. 4A). A sterile microscope slide was submerged in 25 ml of medium (M9 or 0.1 LB) in a 50-ml conical tube and incubated at 37°C in an orbital shaker at 100 rpm. To grow the biofilm, the bioreactor was inoculated with 50 ul of overnight culture; the slides were transferred daily to tubes with fresh medium. The nonattached cells were washed off by submersion in saline. The slides were then submerged in saline in a petri plate, and analyzed by confocal microscopy.

Hope it helps!

Hi,

I used to work with P. aeruginosa biofilms and I removed the media by pipetting but I didn't disrupt it. I don't know what you're trying to do but I think that biofilms are well attached to the wells so you should just pipet the media softly to not disrupt it. You place your pipet against the wall of the wells and aspire the media softly without moving your pipet abruptly. And we used Greiner Cellstar too, I don't know if the label has something to do with the attachment of the biofilm but just in case... And yes, 24 well plates are more suitable for that type of assay

Hannah Dziura Ideally, all I want to do is generated PAO1 biofilms in the bottom (flat) of 96 well plates and be able to remove the media. The problem I've been having is that the growth is so sticky that it all comes off in one big string. I have tried changing the media to a more defined one (MOPS medium), but this doesn't appear to grow enough. I plan on adding some different carbon sources to try and optimise growth

Jonathan ...... are you using high protein bind 96 well plates, perhaps this may help

Hi,

Another way to growh biofilm is using the Calgary device or also called the MBEC assay. It is more use for antibiotic suceptibility test, but you can growth biofilm whitout disruption.