I'm doing a biofilm assay with streptococcus mitis and mutans. I get excellent biofilm formation after 48-72hr hours incubation (rather than standard 24Hr), but i am worried that this biofilm would actually be dead cells depositing on the bottom instead.....can someone please advise me for this assay?
For Streptococcus biofilms, the best time periods are between 48-72 h depending on how thick you want your biofilm to become. As mentioned by Misagh, it is best if you have an initial adhesion step and wash out non-adhered bacteria in order to remove planktonic bacteria which would be more susceptible to death during long incubation times. The optimum incubation time will be determined based on your initial adhesion seeding (how much bacteria you allow to settle on your surface), the availability of nutrients throughout the incubation process (i.e. fresh media every 12 or 24 h), and the desired thickness of the biofilm.
As for your comment about whether dead bacteria sink faster, I have done a pilot study to help confirm a hypothesis in one of my manuscripts "Environmental and centrifugal factors influencing the visco-elastic properties of oral biofilms in vitro". In this case, surface damage prevented bacteria from coaggregating, thus slowing the settling in media at room temperature. With dead bacteria, depending on method of cell death, the cellular materials from the dead cells could aid in coaggregation of live cells giving you a mixture settling at the bottom, heavier than that of only live cells coaggregating.
Biofilm formation is a complex and active process. Attachment proteins and secretion of various biofilm components (ex. polysaccharides) is an integral aspect of biofilm formation. Since it takes an active process to form biofilm, I don't think deposition of dead cells would be forming biofilm. Dead cells would likely stay planktonic, or at the most, very loosely adhered to the bottom. After biofilm formation, washing the vessel in which you are performing the assay at least once should remove any loosely bound (and dead) cells. In conclusion, to answer your general question, I don't think dead cells would alter your results significantly, even if the dead cells do sink to the bottom.
I hope this helps.
I agree with above, wash the cells. Actually, it is better to let bacteria attach for a few hours, then wash and allow only adhered bacteria to form biofilms. Dead bacteria will always be present , but not as much as you think
Thanks guys for your help. Would you recommend me to investigate biofilm formation after 48 or 72 hours?
I'm not experienced with Streptococcus, but with Vibrio I have seen anywhere from 6 hours up to 72 hours. I'm not sure if this will be of any use to you, but maybe you can do 6, 24, 48 and 72 hours for each strain or treatment or whatever it is you are testing. It will give you a nice time course. Could be useful.
For Streptococcus biofilms, the best time periods are between 48-72 h depending on how thick you want your biofilm to become. As mentioned by Misagh, it is best if you have an initial adhesion step and wash out non-adhered bacteria in order to remove planktonic bacteria which would be more susceptible to death during long incubation times. The optimum incubation time will be determined based on your initial adhesion seeding (how much bacteria you allow to settle on your surface), the availability of nutrients throughout the incubation process (i.e. fresh media every 12 or 24 h), and the desired thickness of the biofilm.
As for your comment about whether dead bacteria sink faster, I have done a pilot study to help confirm a hypothesis in one of my manuscripts "Environmental and centrifugal factors influencing the visco-elastic properties of oral biofilms in vitro". In this case, surface damage prevented bacteria from coaggregating, thus slowing the settling in media at room temperature. With dead bacteria, depending on method of cell death, the cellular materials from the dead cells could aid in coaggregation of live cells giving you a mixture settling at the bottom, heavier than that of only live cells coaggregating.
If your planktonic growth is fast, dead bacteria may deposit and affect your biofilm and its formation. Depending on what you aim to achieve, you could seed your surface and then change the medium frequently, based on the planktonic growth of your bacteria. Again, what you should do depends on what you aim to use your biofilm for.
As Mesrop said that Biofilm formation is a complex and active process, cell death considered and observed during biofilm formation, and thought to be a part of the biofilm developmental process.
(The death and lysis of microbial cells leads to the release of cytoplasmic
contents, many of which are rapidly degraded by enzymes. However, some
macromolecules survive intact and find new functions in the extracellular
environment), this is part of the abstract of this interesting review paper.
http://www.ncbi.nlm.nih.gov/pubmed/23848166
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