Hi i am trying to compere biofilm producing of different staphylococci isolates. But unfortunately with no luck. i followed this protocol: Cultures diluted 1:200 in TSB with 0.25% glucose and inoculate in 96 nunclonDelta plate (Tissue culture plates). plates incubate for 24h or 48h at 37C. plates were washed three times with PBS. incubate for 1 hour at 60C. stained with crystal violet follow 15min room temperature incubation. washed three times with deionized water and dried in an inverted position. after drying plates were read with microplate reader. we add two controls, PC-ATCC 35984, NC- ATCC 12228. non produced biofilm. help will happily be accepted .
Hii Tamar Leshem ,
The protocol you are following is being used almost everywhere. Although you can modify/add few steps.
Before giving wash to the biofilm, you can fix it with 99% methanol (100-200 micro lit.).
Then after staining, if the absorbance reads are much higher, then you can wash excess crystal violet using 33% acetic acid (150-200 micro lit.).
You can refer following article which is having comparison of six different methods for biofilm quantification.
Peeters, E., Nelis, H. J., & Coenye, T. (2008). Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. Journal of microbiological methods, 72(2), 157-165.
Best wishes !!!
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