@Klas Udekwu. You've got a point. The composition of biofilm matrix is very complex and various molecules are expressed at the different environmental conditons. In standard laboratory settings S.aureus and S.epidermidis strains often are CR-positive (PNAG-positive) and then they switch into Embp or Bap/Bhp matrix mode and so on...

Congo red is an out dated indication of an organisms capacity to form a biofilm that we now know is not suitable for that. There are many reasons for this including the fact that staph can make biofilms using protein (and eDNA) even if they don't produce polysaccharide. So even if the staph is ica negative it might still make a biofilm. I don't know how you are determining if your bugs are ica positive or negative but I guess you are using some form of PCR. If so then one more thing you need to look at is if an insertion element (eg IS256) has jumped into one of the ica genes so it no longer produces polysaccharide. The only sure way to determine if your bugs produce a biofilm is to do the biofilm assay.

Yes it is an outdated method for biofilm quantification, however some researchers use it to screen biofilm- +ve strains at large scale. Reason is pretty same as Dr. Sean explained. To quantify biofilm you can use simple crystal violet assay. Moreover, in your case if you are detecting ica +ve or -ve based on PCR. PCR results could be flawed and and you may end up with false negatives. Better you go with dot blotting or southern blotting to identify ICA +ves.

Biofilm asssay with crystal violet can help to solve your problem for quantification. As i know, method is called semi-quantitative but it works well. You can only estimate your result as percentages in increase or decrease in biomass.

As said previously, congo-red is targeting only one class of characters that allow for biofilm formation. Unfortunately, some strains will use other characteristics to grow in biofilms. Congo-red is reliable but has limited uses.

Crystal Violet is not as specific and will bind indistinctly to bacteria, thus showing biofilm formation without taking into account the characteristics which the bacteria are using to form it. It is a great way to visually identify differences in biofilm thickness for example.

Based on what you said, I think Crystal Violet will be a more reliable method than congo-red to detect biofilms at a macro level in your case.

Congo red agar (CRA) screening is not useful as a qualitative screening method for biofilm formation.The combination of CRA and microplate and CLSM methods provided a better prediction of the presence of ica genes and biofilm formation in S. aureus isolates.

Your results confirm what many researchers have observed over the years which make Congo Red Agar screening unsuitable to detect all biofilm formation. Crystal Violet Staining in microplate provides a better correlate of biofilm formation in the macroscale.

I have recently quantified the biomass of bacteria in biofilms using S. aureus and E.coli and they worked well. I am yet to try Congo red agar but I have also tried slime production using 0.5% safranin and it work well with S. aureus and E. coli biofilms cultured overnight is LB broth.. Safranin staining on the walls and bottom of the tubes showed adhesion.

Do You know to which polisaccharide of S.aureus slime Congo Red bind to?

Dear Junka congo red is a well known Amyloid binding dye and its is reported that S. aureus producing Amyloid for biofilm formation. For more detail red the article: Functional Amyloids Composed of Phenol Soluble Modulins Stabilize Staphylococcus aureus Biofilms:

Kelly Schwartz,

Adnan K. Syed,

Rachel E. Stephenson,

Alexander H. Rickard,

Blaise R. Boles.

PLoS Pathog 8(6): e1002744. doi:10.1371/journal.ppat.1002744.

Dear Dr Mirani, thanks for this article. It seems that S.aureus biofilm matrix composition is far more complex than we originally thought. This matter is quite interesting, as CR is well-known from ability to bind to amyloid and it has been used in histology for, I don't know, 30 years or something. Also CR microbiological assay is used for many years in microbiology. And the presence of amyloid in S.aureus was discovered just like a year ago:)

I do not think Congo Red agar method is reliable for detection of biofilm in Staphylococci. Using PCR, we investigated a number of Staphylococcus isolates that were positive for biofilm formation on CR agar and none of the isolates was positive for genes (bap,icaA, icaB, icaC, icaD, icaR) coding biofilm formation.

@Klas Udekwu. You've got a point. The composition of biofilm matrix is very complex and various molecules are expressed at the different environmental conditons. In standard laboratory settings S.aureus and S.epidermidis strains often are CR-positive (PNAG-positive) and then they switch into Embp or Bap/Bhp matrix mode and so on...

In S. aureus in many strains PIA/PNSG expression is suppressed and must be stimulated. Thereby, different stimuli are necessary for different strains. In S. epidermidis it was demonstrated that isogenic mutants of the icaADBC displayed red colonies on CRA compared to the black and dry colonies of the wildtype. Therefore, it is assumed that CR binds to PIA/PNAG.

CRA agar is usefull to screen for subpopulations/single strains with a phenotypic switch. However, morphology on CRA can not be used to determine biofilm formation.

Formation of biofilm is a process which is highly stimulated at stress conditions. More than 99% of bacteria lives in a biofilm. 1% is the planktonic ones which can be seen in the bulk water phase. While doing Congo red plates, you are actually checking the production of slime. And indeed the black coloration is due to the presence of curlie fibers which helps the formation of exo polysaccharides! Among all other bacterial fibers, curlie is distinguishable as it has the property to bind with congo Red. Here by CR assay we are utilizing this property. And its true that if the bacteria is having curlie fibers, it has the capability to produce slime. In a multi plate of 96 wells, bacteria is more or less stressed. so if it has the capacity to produce biofilm, this happens as they are under stress. So CR plate  assays are definitely worth in most cases.

I agreed with all these answers .

Thank for all your helpfulness .  

CRA is not reliable technique to biofilm detection.

Hi, Zulfiqar,

CRA is not a reliable method for biofilm detection. You can check this article for more information:

http://www.scielo.br/pdf/bjid/v15n4/v15n4a02.pdf

Best,

Arizaldo