Dear Bassem B. Halla

The following paper help you to assess the antibiofilm activity through crystal violet method.

At the last step before taking the OD you should dissolve the absorbed Crystal Violet (in control and test) in a fix volume of solvent which will developed on the metal surface and than after you take the OD.

See in paper Zaborina et al., Annals of Clinical Microbiology and Antimicrobials, 2006

Biofilm formation.   P. aeruginosa strains were grown in

96-well plates in M63 supplemented with 0.5% casamino

acids and 0.2% glucose. Plates were incubated at 37°C

under mild shaking at 50 rpm (C24 Incubator Shaker,

New Brunswick Scientific, Edison, NJ) for 8 hrs. The wells

were then rinsed thoroughly with water and the attached

material was stained with 0.1% crystal violet, washed with

water, and solubilized in ethanol. Solubilized fractions

were collected and absorbance measured at 550 nm with

a Plate Reader.

Best

Olga

Hi  Bassem,

I have done a similar assay for bacteria grown in liquid medium.

You can find the details in my reference by Chandra  H et al.

Microbiology. 2010 Dec;156(Pt 12):3669-77. doi: 10.1099/mic.0.043828-0. Epub 2010 Aug 19

If you have any questions please let me know.

Hope it helps

Thanks

Harish

Dear Bassem,

I used a protocol described in this paper to run my biofilm assays.

Merritt, J. H., Kadouri, D. E., & O’Toole, G. A. (2005). Growing and Analyzing Static Biofilms. Current Protocols in Microbiology, 0 1, Unit–1B.1. http://doi.org/10.1002/9780471729259.mc01b01s00

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568995/

This paper also has a protocol that I have attempted.

https://www.nature.com/articles/s41598-018-27193-8

1- grow the bacteria 2- guarantee the pure culture 3- Next form the biofime, add 500 microLiters of bacteria into a 24 well microplate. 4- keep without no agitation for 24 or 48 or 72 days until biofilm forms 5 - Then centrifuge with microplate rotor 6- discard the supernatant 7-Wash the plate carefully with buffer solution, and air dry 8-Biofilm cells adhered to the well were fixed by the addition of 1000 microliters methanol for 15 min (4 ° C) and then the methanol was removed and air dried at room temperature. 9-Subsequently, added 1000 microliters of 0.1% violet crystal for 15 min. After this time, the plates were washed and air dried again. 10- Then, 1000 microliters of ethanol (96%) were added to each well and allowed to act for 5 min. Finally, 200 microliters from each well will be transferred to a 96-well microplate. The fluorescence intensity of the violet crystal was measured by a microplate reader using a wavelength of 595nm. doubts, [email protected]

I would like to ask that after reading absorbance how can we actually quantify the EPS or biofilm. is it by putting absorbance in certain formula or we need to make standard curve for crystal violet, if yes then how..?