Many research articles have mentioned biovolume as an important biofilm analysis parameter. So, I would like to know whether a higher biovolume represents a thicker biofilm with more number of cells? Or is it a parameter to define the compactness of biofilms? Please reply.
Pradeep - great question. I've most frequently encountered biovolume as a measurement taken from confocal microscopy 3D 'z-stacks' using software such as comstat. Like any image processing measurement, you need to be sure and understand how the software is arriving at its result. Often times, the lower bounds of the volume is the surface of the coupon on which the biofilm is being grown. The 'walls' of the volume are often the lateral edges of the image stack. The roof of the volume is derived through image processing to be where the stain being used to visualize the biofilm is below some determined threshold. Voxels above that threshold are considered within the biofilm, voxels below are considered outside.
Things to be thoughtful of include the following:
1) Depending on how the microscope is set up, the deepest focal plane that can be acquired is sometimes still within the volume of the biofilm. That will leave you with an erroneous estimate of the roof of the biofilm. In such cases, there are no visualizable organisms or matrix because of the optical limitations of the instrument, rather than the top of the biofilm actually being established.
2) Remember that volume is derived from fluorescently labeled voxels and doesn't absolutely correspond to the number of microorganisms or the amount of matrix present. It should reflect in general the proliferation of the biofilm, but be careful concluding anything about the cellularity, 'looseness' or 'tightness' of the matrix, etc.
3) There are more formal ways of addressing some of the ideas that you mentioned, such as 'compactness,' that rely on establishing an {x,y,z} coordinate for every bacterium within the sampled volume, then conducting a series of spatial statistics on that population.
Hope that at least gets your started.
Best, John
www.akadeum.com
Hi Dr. Younger
Thank you very much for your valuable suggestions. I will calculate the biovolume for the biofilms accordingly.
Some articles refer to the bio-volume as total biofilm biomass. This is because not only are the number of cells important in biofilm formation but also the extracellular matrix which can be comprised of proteins, polysaccharides and DNA. Some routine biofilm stains like crystal violet will bind to the biofilm cells and the extracellular martix which is not always the case with fluorescent dyes.
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