Hi.
Im looking for some help. Im working on a Bacillus strain that forms Bio-film, it is growing extremely well reaching a high OD in a 5L Fermenter, however I am struggling to quantify it. Im doing TVCs however the biofilm is making it really difficult. Ive tried spread plate and pour plate using TSA agar.
Has anyone any suggestions.
Thanks in advance
I agree with Bo. Quantitation of biofilm bacteria is very difficult and considerable effort is needed to even approximate the real "CFU" as they retain clump formation. Sonication is the best method in our experience, but it depends on accessibility of the parts of the fermenter where the biofilm is growing. Alternatively you could research a non-lethal detachment method (surfactants etc) in the literature for Bacillus spp
Hello Bo,
you can use the sonication method that will break the EPS matrix and release the bacteria that you can mark (eg SYBR GREEN) and enumerate by epifluorescence microscopy. you just have to know exactly the surface sonicated to know the number of bacteria / cm².
You can find more explication in this publication: Article Assessment of a low-frequency ultrasound device on preventio...
Good luck
Dear Anne Mc Meel ,
I agree with Roger Bayston . Sonication is one of the common tools used to detach biofilms from a surface ( Article An efficient biofilm removal method for bacterial cells expo...
) but also break up flocs (Article Aggregation in Azospirillum brasilense: Effects of chemical ...
). Caution is needed on the settings of the sonicator (amplitude, frequency) and time to avoid disrupting the cell well. Burdman et al. (1998) also observed immediate re-aggregation of cells once sonication was stopped. Interestingly, 0.2 to 0.5 M urea was also effective in disrupting microbial aggregates.Other methods you can try include homogenisation using a rotating homogeniser. Rizzi et al. (2006) resuspended detached biofilm in PBS and homogenised the solution 6x at 8000-24000 rpm ( Article Response of methanogen populations to organic load increase ...
).Dunsmore et al. (2003) used vortexing for 2 min to disperse cell clusters ( Article A novel approach to investigate biofilm accumulation and bac...
). Gagnon and Slawson (1999) compared three methods to remove cells from a surface (scraping with a utility knife, swabbing and stomaching) and 4 methods for cell resuspension (tissue blender, vortex, stomacher, sonicator). The best overall method was found to be stomaching.In order to know whether a particular method was successful in breaking up the flocs one can use the method suggested by Zineb Fechtali Moute . In order to establish whether cells are viable (metabolically active) or not one can try the CTC stain ( Article A New Direct Microscopy Based Method for Evaluating in-Situ ...
).Last but not least here is a link to a website in which various mechanical homogenisation techniques are listed and discussed: https://homogenizers.net/blogs/blog/choosing-a-homogenizer-technology
Hope this helps?
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