RELATIONSHIP BETWEEN TWO TECHNIQUES FOR BIOFILM RESEARCH
Arce Miranda JE, Baronetti J, Albesa I, Paraje MG#
Departamento de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba
Haya de la Torre y Medina Allende, Córdoba (Argentina), CP: 5000.
Introduction
Biofilms are microbial communities of surface-attached cells embedded in a self-produced extracellular polymeric matrix. They can cause significant problems in many areas, both in medical devices (e.g. persistent and recurrent infections, device-related infections) and in non-medical ones (1), therefore its detection and quantification is very important. Two methods used to biofilms evaluation are crystal violet (CV) and confocal scanning laser microscopy (CSLM); however a correlation between these two methods has not previously been reported. In this work, a comparative study on correlation between them, evaluated under different culture conditions of biofilms growth previously assayed in our laboratory (2), was realized.
Materials and methods
In this study, the biofilms formation of the reference strain of S.aureus (ATCC 29213) was evaluated by CV and CSLM.
Quantification by CV
The biofilms-forming ability was measured by determination of the adhesion to polystyrene microtiter plates (3), which is based on the ability of bacteria to form biofilms on solid surfaces and uses CV to stain biofilms. Briefly, a final cell concentration of approximately 1×109 cfu/ml of S.aureus was put with Triptyc Soy Broth (TSB) into each well of flat-bottomed microtiter plates, with/without supplements and incubated at 37°C for 24 h. A quantitative assessment of the biofilms formation was obtained by extracting the CV with a bleaching solution. The Biofilms Biomass Unit (BBU) was arbitrarily defined with 0.1 OD595 equal to 1 BBU.
Biofilms Research by CSLM
Biofilms were observed by CSLM. Prior to imaging, the samples were rinsed with sterile potassium phosphate buffer for 10 min and were then stained with propidium iodide to detect bacterial cells. After being washed in PBS, the sections were incubated with fluorescein isothiocyanate-concanavalin A conjugated to stain the glycocalyx matrix. Then, images of the vertical (X/Z) sections from three randomly selected positions were obtained and analyzed using an Olympus Fluoview FV 1000 (4).
Results
Previous studies in our laboratory have demonstrated that the capacity of S.aureus to form biofilms changed under different culture conditions. With the information obtained from the two methods employed (table), a high correlation between them (R2 = 0.940), calculated by linear regression, was found.
Conclusions
Our results showed a good correlation between the two techniques, which were assayed at different culture conditions of biofilms formation. The quantification by CSLM was able to detect change in the biofilms formation of S. aureus, for example high levels of growth were observed in presence of Glucose or NaCl&Glucose. In addition, this method allows us to obtain data on biofilms structure (3D visualization) and the differentiation between live and dead bacterial cells. However, training personnel and confocal microscope are necessary for the development of this technique. In the same way, although is not possible to obtain biofilms structure information by CV staining, this assay, simple and cheap, also was sufficiently sensitive to evaluate the ability of this bacterium to form biofilms. Therefore, since similar results were obtained by these two methods, they could be indistinctly used for the study of biofilms formation on polymeric surface of medical devices.
Acknowledgments
The authors wish to thank Drs. C.Mas and C.Sampedro. This work was supported by the following Grants: FONCyT, CONICET and SECyT.
References
1-Peeters E, Nelis HJ, Coenye T. Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J.Microbiol.Methods. 2008; 72:157-165.
2-Arce Miranda JE, Sotomayor CE, Albesa I, Paraje MG. Biofilm formation and cellular stress in Staphylococcus aureus under different culture conditions. 2010. Under revision.
3-O´Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol.Microbiol. 1998; 28:449?461.
4-Zernotti ME, Angel Villegas N, Roques Revol M, Baena-Cagnani CE, Arce Miranda JE, Paredes ME, et al. Bacterial biofilm evidence in nasal polyposis. J Investig Allergol Clin Immunol. 2010. In press.
