Sorry I have a very simple and basic question. Now I am screening the ability for S. aureus and S. epidermidis to form biofilm in 96-well plates. The media is TSB with 0.25% glucose. After 24h culture, I found one strain of S. epidermidis form biofilm at the air-liquid interface on the well. However, I also found some strains form at the bottom of the well. So I am a little confused, does S.aureus and S.epidermidis form biofilm at the air-liquid interface in 96-well plates (just like P.aeruginosa), or they can also form biofilm at the bottom.
Thank you very much.
When grown in liquid culture without rotation, many bacterial pathogens, including S. aureus, form biofilms on both solid surfaces or at liquid-air interfaces.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795244/
This may can help you,
http://www.scielo.br/pdf/bjid/v15n4/v15n4a02.pdf
In Staph, this is strain dependent. Also, the assay is influenced by culture conditions.
May I suggest the following reference?
Biofilm Localization in the Vertical Wall of Shaking 96-Well Plates
Luciana C. Gomes, Joana M. R. Moreira, Manuel Simões, Luís F. Melo, and Filipe J. Mergulhão*
LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal
*Filipe J. Mergulhão: Email: tp.pu.ef@mepilif
In my experience, using 96-well plates, biofilm growth tends to occur to a greater extent on solid surfaces. However, the degree of biofilm formation varies considerably depending on the isolate.
I agree with all of the comments above. Biofilm formation will be influenced by the isolate/strain, by the surface, by the culture conditions (media, rotation vs stationary). I would also agree that surfaces of the 96-well plate wells are best for biofilm formation, in fact, by definition the term 'biofilm' normally refers to microbial growth that is attached to a surface and encased in a self-produced matrix. The type of surface material has a large influence on biofilm formation. Not all microorganisms are good at forming biofilms on polystyrene. Perhaps one or more of your isolate(s) are not particularly good at forming biofilms on polystyrene under the conditions you are using.
Staphylococcus is quite a broad genus and encompasses various diverse strains.
If you are just screening for biofilm formers you can choose the species that forms biofilms attached to the plate. The biofilms that are not attached or float would probably be more difficult to manipulate ! The biofilms that float may be interesting because they could contain biofilm amyloids which some biofilm species express at certain stages of maturity but i haven't really seen that many in Staphylococcus.
To promote more biofilm growth in Staphylococcal species , high bind (ELISA type) plates or just Hi-bind plates are recommended. These have a specially modified surface for binding organic molecules . Lower grades of plastic do not cultivate biofilms well.
Not all species of Staphylococcus will form biofilms at 37C but if you lower the temperature to 20 C you might discover some more biofilm formers .
My optimal biofilm cultivation technique is to grow the bacteria to stationary phase, usually overnight, then dilute the suspension 1/100 with dilute media. The media is usually 1/2 strength TSB or 1/5 in the case of environmental isolates. Lastly it is very rare that I shake or rotate the 96 well plates. For S. aureus, I would incubate for 3 days at 20-25C (cover with aluminium foil , make sure plates are on a flat surface and then quickly stain with crystal violet in the procedure described by McGloughlan I think .
good luck with your screen .
some of the procedures are outlined here
http://link.springer.com/article/10.1007/s00253-014-6169-6
and specific for staphylococcus here
http://pubs.acs.org/doi/abs/10.1021/pr200029r
Biofilm formation in S. aureus or S. epidermidis is strain specific. Bacterial attachment/adhesion is seen in air-liquid interface almost all strains. However, the biofilm layer is formed on bottom surface of 96-well F-bottom plates. Sugar type and concentration also affect biofilm formation level in staphylococci.
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