I am currently investigating biofilm formation in Salmonella isolates using 96-well crystal violet assays. The protocol I follow utilizes multichannel pipettes (opposed to other rinsing and removal methods I have read about others using). From 1:100 diluted overnight broth plates are inoculated and incubated (at various temperatures), broths are removed after and wells are washed using MQH2O until clear. I then stain using 0.1% CV for approx. 15mins (using a higher volume than the broth; e.g. 100ul broth and 125ul 0.1% CV), then washing wells once more after. after drying for 48h or more I solubilize the dye using 30% acetic acid and record the OD600. when comparing the results to a blank negative control of 30% acetic acid (approx. 0.05 OD600) calculations are showing very high biofilm formation (high OD600 in contrast to the negative control) when the lab isolates are supposed to have for example low formation or even none. I suspect there is insufficient pipetting of planktonic cells/ CV leaving a coating which is skewing OD600 measurements. This issue is consistent across most replicates and temperatures. I would love to hear any insight, thank you.
DEar Aaron Turner
i worked with biofilm many years ago and i never worked with Salmonella but from my experience with other strains there are some point of your protocioll that can be optimized to improve the reprodubility and be sure that the biomass that you measure with CV is biofilm and not just died planctonic bacteria.
1) In my experience if yuo perform biofilm formation in microtiter plates (with bacteria adesion on the plate bottom) and not with PINS, to limit the effect of asepcific adesion due to the gravity and bacterial death, is better to perform a media exchange step after some our of adesion to remove the excess of plactonic non aderent bacteira and give to the biofilm formers more nutrients.
You can see an example of it in the paper that we pubblished for GBS
https://journals.asm.org/doi/10.1128/AEM.03627-13?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed
2) You can use the multichannel to fill the wells by pipetting in the well border to limit but i think that it is preferable remove the media by vacuum mainfold or by reverting the plate.
3)Once you removed the broth, i suggest to you to perform at least 3 washing (i used also PBS instead water to maintain good viability) to be sure that you removed all the non aderent cells.
good luck
manuele
Aaron Turner Hi, I have recently checked biofilm forming ability in E. coli using CV assay. This paper will help you:
Article Prevalence and mechanisms of antibiotic resistance in Escher...
I have always used water to rinse but the rinse method is important so that you do not lose biofilm in the rinsing. You do not give the readings, and you do not say how long you incubate the plates, but accepting Dr Turner's points about gravitation effects and dead planktonic bacteria, it is quite possible that the strains that you are testing actually do give higher biofilm readings than the controls (what are the controls? Sometimes lab-maintained control strains behave sub optimally). I also favour vacuum extraction.
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