In bacteria biofilm, a lot of bacteria become metabolically dormant while some are still actively growing. How do you separate them?
Staining for metabolically active bacteria or flow cytometry for sorting out metabolically active bacteria
How can you pass a biofilm through a FACS ?
Or else, how can you resuspend a bacterial biofilm into a homogeneous solution ?
Yes, Joseph. You raised a critical point. Flowcytometer might be a good approach but the question is how to detach bacteria from biofilm without damaging them. What dyes can we use to tell which bacteria is active or dormant.
beside stains that will color cells based on membrane integrity, such as Invitrogen LiveDead, I don't know much about microscopy sorry. I'm though very much interested by the topic.
I'm though thinking about somthing like FISH for proteins ? Targeting specifically ribosomal protein for example. If fluorescence signal is shown to increase proportionally with ribosome presence, this could be a nice indicator of metabolic activity. Ribosome concentration in cytoplasm is indeed a good proxy of metabolic state of a bacterial cell.
Still the question of biofilm structure, how you fix sample and how you permeabilize cells so your probes can find their target (that is they shouldn't be denaturated) seems a considerable challenge!
Hi everyone, it all depends on what you want to do. You asked a broad question. Be more specific. You want to study them live or fixed? Distinguish attached to a surface? What type of biofilm? Use after distinguishing? I can go on and on...
Hi everyone, it all depends on what you want to do. You asked a broad question. Be more specific. You want to study them live or fixed? Distinguish attached to a surface? What type of biofilm? Use after distinguishing? I can go on and on...
What about the detection of SCV (Small Colony Variants) using auxotrophy test?? Could be also an idea to identify dormant or less metabolic active bacteria !!
I am thinking whether some metabolic indicators would help, such as Tetrazolium-based dyes including XTT, TTC, and CTC.
Tetrazolium dye reduction depends on NAD(P)H-dependent oxidoreductase enzymes in cells. They are soluble and mainly colorless and can be converted to fluorescent or colorimetric compounds by metabolically active bacteria.
Dear Daniel
you can go for vital staining with Invitogen Live/Dead and scan those with confocal laser scanning microscope with disturbing any film or removing in situ bacteria. 2D or 3D scanning is possible with CLSM.
Divya, I am interested in dormant bacteria but not dead or membrane-damaged bacteria.
use these papers:
Hammes, F., Berney, M., Egli, T. (2011): Cultivation-independent assessment of bacterial viability. Adv. Biochem. Engin./Biotechnol. 124, 123-150
Rochelle, P.A., Camper, A.K., Nocker, A., Burr, M. (2011): Are they alive? Detection of viable organisms and functional gene expression using molecular techniques. In: Sen, K., Ashbolt, N. (eds.): Environmental Microbiology. Caister Acad. Press, Norfolk, UK, 179-202
Dr. Flemming, I would like to have a look at those papers. Please send me PDFs to [email protected]. Thank you very much.
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