Hi all,
I am working on Pseudomonas sp. I am trying to explicate the ability of the bacteria to form biofilm. So please help me to find a good methodology to find the same.
Thanking you in advance!
Dear Mr. Muneeb,
The formation of bacterial biofilm can be quantified by using crystal violet assay or XTT reduction assay. We can perform this assay in test tubes or poly styrene coated plates (96, 48, 24 or 6 well plates). Here i am attaching 2 papers which may useful for your work. Please find the attachments.
All the best.
The above mentioned are useful and standard methods. I am following them for few years and results are good. I do with test tubes and crystal violet. Good for screening.
A modified crystal violet assay can be performed for visualising the biofilm formation.
Detailed Methodology (taken from a published manuscript)
Biofilm formation was achieved by aliquoting 100 µl (1 x 105 CFU/ml) of microbial culture into a 96-well microtitre plate, followed by incubation for 24 hr at 37°C. Biofilm biomass was assessed using the modified crystal violet (CV) assay. The overnight incubated plates were washed thrice with 100 µl of PBS to remove loosely attached cells. The plates were then air-dried/ and oven-dried at 60°C for 15 min. Subsequently, cells were fixed with 200 µl of methanol for 10 min. Excess methanol was drained and the plate was air dried. The plate was stained with 100 µl of 0.1% crystal violet and incubated at room temperature for 5 min after which the plates were washed three times with PBS to remove unabsorbed stain. Destaining was performed by adding 200 µl of ethanol (95%). 100 µl of the destaining solution was then transferred to a new plate, and the absorbance was determined at 595 nm using a microplate reader (Universal microplate reader ELX 800; Biotek instruments Inc., Winooski, USA).
Percentage Biofilm formation = (O.D. Maximum adherence - O.D. Blank) / O.D. Maximum adherence * 100
Those methods work well and are well documented. Another method for looking at the formation and structure of the biofilms is to use confocal microscopy. We transformed the bacteria with a plasmid to produce GFP. You can then capture the size and structure of the biofilm while it is growing without interruption. Depending on your setup, you can image the change over time and have a 3D image/model of your biofilm. Whichever way you use will depend on your facilities and end goals.
Best of luck
There are many methods out there, so I think it also depends on the kind of time, budget and facilities available to you and also the kind of results you want - qualitative or quantitative or both. You can try putting the keywords in your literature search as Pseudomonas biofilms or even your specific strain. There may be direct method-related papers or other susceptibility based study papers, from which you can extract methods from. Then you can identify the method that best fits your agenda, further modify and optimize it to get quality results. Good Luck!
Just check for publications with following key words: Micro-titer Plate Assay for biofilm formation, as well as Congo Red Agar for slime production. Your will get a lot of papers with perfect methodologies. Check this link for a video too.
http://www.jove.com/index/Details.stp?ID=2437
This paper -with video really helps!!!
Regards,
Crystal violet staining after growing your biofilm on the appropriate surface is probably one of the quickest easiest methods. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/ and also this has other methods
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1413-86702011000400002
Hello Muneeb,
You can use this simple method:
Article Synthetic DNA molecules in biosensing of biofilms
Good luck,
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