I think biofilm removal from liquid, membrane filtration is the only option.

Dear Andrea,

Although I work with pseudomonads, we often have the issue when looking at growth rates or biomass production of whether or not to include biofilms in our assays. Like Bacillus, pseudomonads produce air-liquid interface biofilms (or pellicles), and sometimes these are strongly attached at the meniscus to the vial walls, sometimes not, and sometimes they are so delicate that they fall to pieces when you move the vial.

Often when we are looking at growth we decide to measure 'total' growth, i.e., all of the growth in the vial, from sunken biofilm, cells in the liquid column and those in the biofilm at at the top. If this is what you want to measure, then the simplest thing to do is to vigorously mix the vial until all lumps are nbroken up. This can ne very difficult (depending on the biofilm) – you could add some glass balls to help break it apart, or try sucking it in and out of a pipette tip - but what ever method of disruption you choose should not damage the cells (sometimes the problem using a solicitor or adding a detergent). You can get around this problem be defining a very straightforward protocol - 'shake vigorously for two minutes' - and stick to that, so any errors are limited and you are being consistent.

If you want to measure growth or biomass in the liquid column, then we have found that you can often carefully manoeuvre a pipette tip down into the liquid column at the side of the biofilm, and then carefully sample 1 ml or so to make an optical density (OD) measurement or to plate out for cell counts. Clearly, the more robust the biofilm is, the easier it is to avoid sampling that by mistake.

If you want the measure the biofilm itself, then, depending on how robust it is, draw it up using a pipette (say, all visible biofilm, or a 1 ml sample), then vigorously mix it before OD or plating.

In some recent work my PhD student has just published (Jerrdan et al., 2020; FEMS Microbiology Letters, 367, 2020, fnaa187), we found an interesting distribution of cells immediately below our biofilms and down into the liquid column, and were able to show by careful sampling at different depths that cells migrated between the air-liquid biofilm and the lower liquid column to maximise productivity (biomass) in the whole vial.

Andrew

Andrea Carobbi, I agree with Andrew J Spiers; besides, you can gentle sonicate your biofilm containing culture and then measure OD

Thank you all.

Now, the problem here is that my assay is conducted using bacterial O.N. liquid cultures that I normalized to a certain OD which is my "time zero". I have three replicates for each strain. During the assay, I count the number of cells for each strain at determined time points. The number of counted colonies in the three replicates of each strain are coherent, besides for Bacillus samples. Those three never match. I believe is because of the biofilm interfering with the OD measurement.

I'm not interested in biofilm.

I tried to measure growth by collecting a sample below the biofilm but it doesn't work. As Andrew J Spiers mentioned, it might be that there is a difference in the number of cells depending on the depth of sampling.

Sonicating the culture is an option but you won't get rid of the biofilm but only fragmented it and it's likely that it will continue to interfere with the measurement.

Filtering seems the only way. I thought to filter the O.N. liquid cultures before measuring the OD and normalize it.

Finger crossed.

Dear Andreea ,

Try to shake the bacillus subtilis tubes very well using an automatic vortex. An important factor to keep in mind is that the stirring should be the same for all tubes. I would recommend you to count up to 20 each time you shake and in the next 5 seconds to take the sample for densitometer readings. Always try to take half of the tube. You will see that the results will be similar and will be reproduced.

Andrea Carobbi , could you remove biofilm or avoid its formation? I recently encountered a similar problem with the formation of biofilm at the air-liquid interface when cultivating Bacillus subtilis in a multiwell plate. I used sterile glass beads in each well and it worked for me.

Glass beads, as well as flasks with springs (baffled flasks), are widely used for Streptomyces species to obtain dispersed mycelium, and based on this I tried glass beads for B. subtilis.

Hope this helps. Good luck.