If the purpose of the experiment is to determine biofilm disruption properties of a substance/compound, planktonic cells should be removed since they will caused false results. the higher the biofilm disruption, the higher the number of planktonic cells.

Dear Hanna

if you are nor washing away the phlanktonic cells but you centifuging it and in some way you incorporating it in your biofilm, you over estimate the biofilm population and considering that in General treatment are much more effective against planktonic than sessile cells you can also overstimate the efficacy of your treatment because you can see a strong reduction in viability respect the non Treated control sample because you kill all the planktonic but very few biofilm.

for example if your compounf A in 24h of incubation is able to kill 90% of cells planktonic and 10% in biofilm, if you wash away all the planktonic and you start with 100 biofilm cells you will obtain 90 live cells and viability reduction is the 10%, on the contrary of you mantain the plantonick cells, if for example those are 50% of Total cells after centrifugation you will have 200 cells (100 from biofilm and 100 planktonic) and if the treatment kill 90 planktonic and 10 biofilm cells at the end you kill 100 out of 200 that correspond 50% of viability reduction which is much more higher than the Real affect of your compounds on the biofilm.

is it True that in vivo biofilm are different but is important the your in vitro model mantain compactnes, slower metabolism and different oxygenation which are tipical of biofilm and contribute to their higher resistance to drugs, otherwise way use biofilm model and kit test the molecules only against the planktonic form?

good luck

Manuele

Hi Manuele Martinelli - thanks for your response. I understand what you are saying, but surely by pelleting the cells + biofilm, you are pushing all of the cells underneath the biofilm matrix. Therefore, when you administer treatment, it would be absolutely necessary for the treatment to penetrate the biofilm before it would be able to target any cells at all. Wouldn't this in turn be a more impressive result than purely targeting the sessile cells?

If I'm not really understanding it correctly, I'd appreciate any advice you can provide on how to ensure you are targeting the sessile cells rather than the planktonic cells in P. aeruginosa biofilm that predominantly collects at the air/liquid interface.

Hi Hannah

i don not think that centrifugation speed that usually are used to centrifuge bacteria can " pushing all of the cells underneath the biofilm matrix" but just create a double layer and you cannot address which is the layer affected by the treatment.

So if you would like to test a treatment against biofilm , you need to treat a biofilm and not a mixture otherwise while not simply produce layer with centrifuged cells instead lost time and effort to find the right condictions for bacteria adesion.

Moreover is it known that planktonic cells are more active than biofilm so they will convert reazurin with different rate respect than the sessile one, therefore in my opinion at the end of biofilm formation you "HAVE TO" remove all the unattached cells by washing the biofilm with pbs or fresh media and afterwards incubate the biofilm with treatment to determine its susceptibility to it.

i'd like to perform washes to select the real attached cells and dont allow to unattached to compete for the nutrients with them into also during the biofilm formation step and as you can see in fig1 of our paper on gbs biofilm

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993151/

it allows to obtain more mature and thick biofilm.

ciao

Manuele

Manuele Martinelli - excellent response. Thank you so much for taking the time to put your expertise into words for me! :)