For Candida biofilm stainning, a 0.02% crystal violet solution works pretty good. Howerver, for antibiofilm activity I would recomend you to use a metabolic assay because crystal violet will also stain dead cells.

I would only use it for gram-negative bacteria, and only if you remove the stain with EtOH, then you can see biofilm inhibition in a photospectrometer.

No

What strain of bacteria are you talking about?

I would recomend you for metabolic activity assessment, the XTT dye. Because crystal violet only stain all biomass (cells and EPS), is not able to say if the cells are metabolic actives or not.

How about using CLSM and live/dead staining ( e.g. BacLight)?

It has to be checked carefully if this works for your system but in general you get more information than with crystal violet, which just gives you a rough estimate of biofilm biomass (no matter if live or dead, as Taissa already mentioned).

Yes, the Friederike suggestion is also good method.

At the lab, we worked with a 0.1% (w/v) crystal violet solution for quantify P. aeruginosa biofilms…I totally agreed with remarks highlighting that this method is not very precise and I strongly recommand to perform alternative methods in addition.

I am working with the gram +ve strains (Staphylococcus aureus and Micrococcus luteus)

Thanks everybody for your suggestions,

Please let me know, in particular which dye should I use for gram +ve strains (Staphylococcus aureus and Micrococcus luteus)

I think crystal violet will do the job for bacterial biofilm and basically it's the most cost effective method you can use. Good luck

Yes, 1% crystal violet is used to analyze the antibiofilm activity. This method works pretty good but has its own limitation. On a numbe of bacteria Live dead stain may not work due to extracellular DNA in the biofilm.

Dear Gurpreet for quantify biofilm after stain should be resolubilized with DMSO or ethanol and measure absorbance at 570 nm, but for analyze antibiofilm activity el MBEC assay is a reproducible and useful method. With best regards

Thanks Juan..:)

According to my experience crystal violet is better option and i get better results by this then live/dead staining kit.

Hope this helps.. all the best! :)

Thanks Romana..good day

maybe using XTT?

http://www.ncbi.nlm.nih.gov/pubmed/20681967

regards

Thanks guys

Crystal violet (0.1%) is good method if you avoid cross contamination of dye. I am using removal galss coverslip for my experiment, after staining you can remove the coverslip bond bacteria to other plate for the OD measurement. Because in crystal violet plate (may be 24 well plate) bonded stains makes big difference. May be you can try with Invitrogen live/dead stain or may be XTT.

I have tried Live/dead stain as well as XTT but the CV is more easy and satisfactory.

CV staining is easy and commonly used. It is widely applicable to all species. Things to consider are: CV binds to all biomass, not just the microbes, but also slime etc. Also, it does not discriminate between living and dead cells. When doing a biofilm prevention assay (test compound present during biofilm growth) CV stain can be used (keeping in mind that increased slime production will increase your reading. When doing a biofilm susceptibility assay, I prefer to use a metabolic assay (MTT or XTT) or old-fashioned plating (CFU counting). This is ultimately the best way to determine viability of microbes within your biofilm (but elaborate).

For CV stain and MTT assay of Candida biofilms, see the attached paper.

Article Optimized candidal biofilm microtiter assay

Very precise and clearly defined by Bastiaan

In my experiments I use crystal violet 0.1%

Below is a brief description. For more details, see the article

http://www.sciencedirect.com/science/article/pii/S1075996412001230?v=s5

Briefly, bacteria were grown in polystyrene microtiter plates, and the absorbance of incorporated dye (crystal violet 0.01% w/v) by bacterial aggregates, at the optical density 590ŋm, was determined. Thus, the absorbance was equivalent to the density of adherent bacteria.

I would agree with other researchers. 0.1% Crystal violet is better than 1.0 %. I found the protocol of the following article is much reproducible rather than other.

Ref:

O'Toole GA.Microtiter dish biofilm formation assay.J Vis Exp. 2011 Jan 30;(47). pii: 2437. doi: 10.3791/2437.

I agree with Iqbal Jahid.

I just had this paper with methods publised. take a look it may help.

Evaluation of Methods To Assess the Biofilm-Forming Ability of Listeria monocytogenes

http://www.ingentaconnect.com/content/iafp/jfp/2012/00000075/00000008/art00007?token=0051171611fd73e7e41225f40387657476752767025732c74576b34272c5f7b3d6d3f4e4b348be4ae

Can i use crystal violet of gram stain kit for the stainning ??

I wouldn't advise on that. Unless you know the concentration, which is unlikely for most kits.

Crystal Violet is great to see clear differences in biofilm thickness for example. To confirm your results, I will use complementary methods though.

See my pubblication for more information (https://www.researchgate.net/publication/23938962_Nickel_promotes_biofilm_formation_of_curli-proficient_K-12_Escherichia_coli/file/2c2ad22b3afdd7c3e303884c6eca1e35.pdf).

Article Nickel Promotes Biofilm Formation by Escherichia coli K-12 S...

hi researchers , the crystal violet dissolved in Distilled water or in alcohol ?????? to staining the the biofilm .

In water!!

Then resolubilize the attached stain with alcohol 95% (v/v)

0.01% crystal violet for biofilm formation assay gives best results in 96 well microtitre plate. For evaluating metabolic activity in biofilm time point cfu analysis is preferred.

0.01% should indeed be fine and might reduce the background. We prefer 0.01% in our lab. For metabolic activity, CFU is not the assay. CFU screens for living or dead microbes. Metabolic activity in biofilms can be low, but that does not mean they are dead. So for antimicrobial activity, CFU is indeed preferred, for metabolic activity, MTT/XTT/WST assays are preferred because they are rapid. But they do not say anything about dead or alive...

I would agree that metabolic activity detection in bacteria/biofilms by XTT/MTT/CTC assays are preferred. In addition if trypan blue tests are done then anything related to dead or alive may become clear.

U can refer the following article for bacterial biofilm estimation by standard tissue culture plate (TCP) assay (0.1% Crystal violet) articlehttp://www.sciencedirect.com/science/article/pii/S0304389411010843

Hi researcher !! I was wondering that if I can not find the exact OD to measure the quantities of biofilm (OD 590) from CV assay. Can I use the OD620 instead ??? (The organism is Malassezia)

You may use XTT reduction assay, CTC, Resazurine and FDA

I had tried XTT and Crystal violet for Pseudomonas biofilm

I think this link may be helpful

http://www.ncbi.nlm.nih.gov/pubmed/18155789

For staphylococcal biofilm assays 0.4% crystal violet is typically used. Absorbance is usually measured at 492, 570, or 595 nm. Safranin is also used and read at 492 nm.

We prefer performing XTT assays for determining the antimicrobial activity rather than the CV assay. CV assay is good measure for determining the the bacterial capability to form biofilm.

It depends what you are measuring. Crystal violet stains the whole biofilm biomass including cells and extracellular polymeric matrix. The extracellular component can be up to 25% of the biofilm which is important in the quantification of antibiofilm activity. Biofilm = cells + EPS + extracellular DNA . Many other live cell detection methods do not often take the biofilm biomass into account or can stain extracellular DNA. Biofilms are not the same as a collection of planktonic cells. Depending on the biofilm's maturity there can be cells that are viable but not immediately cultivatable and also cells that are senescent so they may appear metabolically inactive. The other advantage of the CV method is that it is very inexpensive and can be repeated many times in order to achieve accurate results . This method can then be validated/correlated using another method mentioned above.

And what about safranin, also used by some researchers?

Hello,

Crystal violet assay has some limitations : not specific of cells or viable cells, you can only detect high range of cells + extracellular matrix (higher than 1E7-1E8, not suitable to follow biofilm formation kinetic); for bacteria like P. aeruginosa you detect biofilm linked to surfaces but also the biofilm present at the air-water interface (according to your biofilm formation model). Viability tests are interesting but you can also quantify bacteria using qPCR and viable qPCR (with a step of extracellular DNA and damaged cells DNA degradation).

I want some information about XTT assay kit for biofilm assay ? Is there anybody use of this method ?

Like Roman Ovchinnikov, I also want to know whether safranin can be used to stain biofilms of pathogenic/non-pathogenic microbes?

Thank you.