Application of FESEM and FTIR for evaluation of Staphylococcus aureus biofilms grown on chitin and polycarbonate membrane

  • R. Baishya
  • R. Chatterjee
  • S. Banerjee
  • M.S. Hasnain
Keywords: Field Emission Scanning Electron (FESEM); Fourier transform infrared spectroscopy (FT-IR); Biofilm; Chitin; Polycarbonate membrane filter; Staphylococcus aureus


The aim of this study was to use Field Emission Scanning Electron (FESEM) and Fourier transform infrared spectroscopy (FTIR), to investigate the structure and the film-forming capacity of Staphylococcus aureus, on two different surface materials, Chitin and Polycarbonate membrane filter. Both the substrates, having diverse properties, showed varied attachments of bacterial and biofilm development. FESEM showed that higher bacterial colonization and biofilm growth was obtained in the rough surface of chitin with complex structure, compared to that of polycarbonate membrane at different time intervals. Due to its high magnification and resolution, FESEM enabled a more detailed analysis of the biofilm cells, influenced by different structural organisations and morphology.  On the other hand, FTIR analyses revealed the chemical conformation of the substrate and its biofilm state. However, the difference in the spectra of the two substrates was not large. Prominent band absorbance at 1200-800 cm-1 for polysaccharides and proteins at 1700- 1400 cm-1 were observed at its biofilm states. These findings promote the prospect of using FESEM and FTIR for understanding biofilm morphology and architecture, as well as its chemical interaction with the substrate.


Anderl, J. N., Franklin, M. J. and Stewart, P. S. (2000).Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrobial Agents and Ahemotherapy 44, 1818-1824.

Aydin Sevinç, B. and Hanley, L. (2010) Antibacterial activity of dental composites containing zinc oxide nanoparticles. Journal of Biomedical Materials Research Part B: Applied Biomaterials 94, 22-31.

Azevedo, N. F., Pacheco, A. P., Keevil, C. W. and Vieira, M. J. (2006). Adhesion of water stressed Helicobacter pylori to abiotic surfaces. Journal of Applied Microbiology101, 718-724.

Banerjee, S., Saha, A., Dutta, S., Baishya, R. and Maiti, P. K. (2015). Effect of different antibiotics against in vitro Staphylococcus aureus biofilm grown on chitin flakes. South Asian Journal of Experimental Biology 5, 22-25.

Barth, A. (2007).Infrared spectroscopy of proteins. Biochimica et Biophysica Acta Bioenergetics 1767, 1073–1101.

Bosch, A., Golowczyc, M. A., Abraham, A. G., Garrote, G. L., De Antoni, G. L. and Yantorno, O. (2006). Rapid discrimination of lactobacilli isolated from kefir grains by FT-IR spectroscopy. International Journal of Food Microbiology 111, 280-287.

Brunner, E., Ehrlich, H., Schupp, P., Hedrich, R., Hunoldt, S., Kammer, M., Machill, S., Paasch, S., Bazhenov, V.V., Kurek, D.V. and Arnold, T. (2009). Chitin-based scaffolds are an integral part of the skeleton of the marine demosponge Ianthella basta. Journal of Structural Biology 168, 539-547.

Chu, P. K., Chen, J. Y., Wang, L. P. and Huang, N. (2002).Plasma-surface modification of biomaterials.Materials Science and Engineering: R: Reports 36, 143-206.

Costerton, J. W. and Stewart, P. S. (2001). Battling biofilms. Scientific American 285, 74-81.

Delille, A., Quiles, F. and Humbert, F. (2007).In situ monitoring of the nascent Pseudomonas fluorescens biofilm response to variations in the dissolved organic carbon level in low-nutrient water by attenuated total reflectance-Fourier transform infrared spectroscopy. Applied Environmental Microbiology 73, 5782-5788.

Fernández-Delgado, M., Duque, Z., Rojas, H., Suárez, P., Contreras, M., García-Amado, M. A. and Alciaturi, C, (2015), Environmental scanning electron microscopy analysis of Proteus mirabilis biofilms grown on chitin and stainless steel. Annals of Microbiology 65, 1401-1409.

Geesey, G. G. and Whit, D. C. (1990).Determination of bacterial growth and activity at solid-liquid interfaces. Annual Review of Microbiology 44, 579-602.

Goodacre, R., Shann B, Gilbert RJ, Timmins E M, McGovern AC, Alsberg K, Kell DB, Logan NA (2000) The Detection of the Dipicolinic Acid Biomarker in Bacillus Spores Using Curie-Point Pyrolysis Mass Spectrometry and Fourier Transform Infrared Spectroscopy. Analytical Chemistry 72, 119–127.

Grenho, L., Jorge Monteiro, F. and Pia Ferraz, M. (2014).In vitro analysis of the antibacterial effect of nanohydroxyapatite–ZnO composites. Journal of Biomedical Materials Research Part A 102, 3726-3733.

Hannig, C., Follo, M., Hellwig, E. and Al-Ahmad, A. (2010).Visualization of adherent micro-organisms using different techniques. Journal of Medical Microbiology 59, 1-7.

Hasnain, M. S., & Nayak, A. K. (2018). Chitosan as responsive polymer for drug delivery applications. In Stimuli Responsive Polymeric Nanocarriers for Drug Delivery Applications, Volume 1. Woodhead Publishing pp. 581-605.

Hasnain, M. S., Guru, P. R., Rishishwar, P., Ali, S., Ansari, M. T., & Nayak, A. K. (2020b). Atenolol-releasing buccal patches made of Dillenia indica L. fruit gum: preparation and ex vivo evaluations. SN Applied Sciences, 2(1), 57.

Hasnain, M. S., Rishishwar, P., & Ali, S. (2017a). Floating-Bioadhesive matrix tablets of hydralazine Hcl made of cashew gum and HPMC K4M. International Journal of Pharmacy & Pharmaceutical Sciences 9(7), 124-129.

Hasnain, M. S., Rishishwar, P., & Ali, S. (2017b). Use of cashew bark exudate gum in the preparation of 4% lidocaine HCL topical gels. International Journal of Pharmacy & Pharmaceutical Sciences 9(8), 146-150.

Hasnain, M. S., Rishishwar, P., Ali, S., Alkahtani, S., Tabish, M., Milivojevic, M. & Nayak, A. K. (2020a). Formulation and ex vivo skin permeation of lidocaine HCl topical gels using dillenia (Dillenia indica L.) fruit gum. Revista Mexicana de Ingeniería Química, 19(3), 1465-1476.

Hasnain, M. S., Rishishwar, P., Rishishwar, S., Ali, S., & Nayak, A. K. (2018b). Extraction and characterization of cashew tree (Anacardium occidentale) gum; use in aceclofenac dental pastes. International journal of biological macromolecules, 116, 1074-1081.

Hasnain, M. S., Rishishwar, P., Rishishwar, S., Ali, S., & Nayak, A. K. (2018a). Isolation and characterization of Linum usitatisimum polysaccharide to prepare mucoadhesive beads of diclofenac sodium. International journal of biological macromolecules, 116, 162-172.

Hetrick, E. M., Schoenfisch, M. H. (2006).Reducing implant-related infections: active release strategies.Chemical Society Reviews35, 780-789.

Hong, K., Sun, S., Tian, W., Chen, G.Q. and Huang, W. (1999).A rapid method for detecting bacterial PHA in intact cells by FT-IR. Applied Microbiology Biotechnology 51, 523–526.

Huang, W. E., Hoppe,r D., Goodacre, R., Beckmann, M., Singer, A. and Draper, J. (2006).Rapid characterization of microbial biodegradation pathways by FT-IR spectroscopy.Journal of Microbiological Methods67, 273-280.

Huang, Y., Leobandung, W., Foss, A. and Peppas, N. A. (2000). Molecular aspects of muco-and bioadhesion: Tethered structures and site-specific surfaces. Journal of Controlled Release65, 63-71.

Jiao, Y, Cody, G. D., Harding, A. K., Wilmes, P., Schrenk, M., Wheeler, K. E., Banfield, J.F. and Thelen, M. P. (2010).Characterization of extracellular polymeric substances from acidophilic microbial biofilms.Appl. Environ. Microbiology76, 2916-2922.

Kurakula, M., Rao, G. K., Kiran, V., Hasnain, M. S., & Nayak, A. K. (2020). Alginate-based hydrogel systems for drug releasing in wound healing. In Alginates in Drug Delivery. Academic Press pp. 323-358.

Lu,Q., Hu, X., Wang, X., Kluge, J.A., Lu, S., Cebe, P. and Kaplan, D.L. (2010). Water-insoluble silk films with silk I structure. Acta Biomaterialia6, 1380–1387.

Milivojevic, M., Pajic-Lijakovic, I., Bugarski, B., Nayak, A. K., & Hasnain, M. S. (2019). Gellan gum in drug delivery applications. Natural Polysaccharides in Drug Delivery and Biomedical Applications, 145-186.

Nayak, A. K., & Hasnain, M. S. (2019a). Plant Polysaccharides-Based Multiple-Unit Systems for Oral Drug Delivery. Springer.

Nayak, A. K., & Hasnain, M. S. (2019b). Background: Multiple Units in Oral Drug Delivery. In Plant Polysaccharides-Based Multiple-Unit Systems for Oral Drug Delivery Springer, Singapore, pp. 1-17.

Nayak, A. K., Mohanta, B. C., Hasnain, M. S., Hoda, M. N., & Tripathi, G. (2020b). Alginate-based scaffolds for drug delivery in tissue engineering. Alginates in Drug Delivery, 359-386.

Norton, T. A., Thompson, R. C., Pope, J, Veltkamp, C. J., Banks, B., Howard, C.V. and Hawkins, S. J. (1998). Using confocal laser scanning microscopy, scanning electron microscopy and phase contrast light microscopy to examine marine biofilms. Aquatic Microbial Ecology16, 199-204.

Parolis, L. A., Parolis, H., Dutton, G. G., Wing, L. and Skura, B, J. (1992).Structure of the glycocalyx polysaccharide of Pseudomonas fragi ATCC 4973.Carbohydrate Research216, 495-504.

Pruzzo, C., Vezzulli, L. and Colwell, R. R. (2008).Global impact of Vibrio cholerae interactions with chitin.Environmental Microbiology10, 1400-1410.

Raad, I., Costerton, W., Sabharwal, U., Sadlowski, M., Anaissie, E. and Bodey, G. P. (1993). Ultrastructural analysis of indwelling vascular catheters: a quantitative relationship between luminal colonization and duration of placement. Journal of Infectious Diseases, 168, 400-407.

Reffuveille, F., Josse, J., Vallé, Q., Mongaret, C. and Gangloff, S.C. (2017).Staphylococcus aureus Biofilms and their Impact on the Medical Field.The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus, IntechOpen, Chapter11.

Rios, P. F., Dodiuk, H., Kenig, S., McCarthy, S. and Dotan, A, (2007),The effect of polymer surface on the wetting and adhesion of liquid systems. Journal of Adhesion Science and Technology21, 227-241.

Sabbatini, S., Conti, C., Orilisi, G., Giorgini, E.(2017). Infrared spectroscopy as a new tool for studying single living cells: Is there a niche? Biomedical Spectroscopy and Imaging6, 85–99.

Samanta, A., De, A., Hasnain, M. S., Bera, H., & Nayak, A. K. (2019). Gum odina as pharmaceutical excipient. In Natural Polysaccharides in Drug Delivery and Biomedical Applications. Academic Press, pp. 327-337).

Sinha, P., Ubaidulla, U., Hasnain, M. S., Nayak, A. K., & Rama, B. (2015). Alginate-okra gum blend beads of diclofenac sodium from aqueous template using ZnSO4 as a cross-linker. International journal of biological macromolecules, 79, 555-563.

Sinha, S.D., Chatterjee, S., Maiti, P.K., Tarafdar, S. and Moulik, S.P. (2017). Evaluation of the role of substrate and albumin on Pseudomonasaeruginosa biofilm morphology through FESEM and FTIR studieson polymeric biomaterials.Progress in Biomaterials6, 27–38.

Steffensen, S, L., Vestergaard, M. H., Groenning, M., Alm, M., Franzyk, H. and Nielsen, H. M. (2015). Sustained prevention of biofilm formation on a novel silicone matrix suitable for medical devices.European Journal of Pharmaceutics and Biopharmaceutics94, 305-311.

Stickler, D., Morris, N., Moreno, M. C. and Sabbuba, N. (1998), Studies on the formation of crystalline bacterial biofilms on urethral catheters. European Journal of Clinical Microbiology and Infectious Diseases17, 649-652.

Stoodley, P., Boyle, J. D., Dodds, I. and Lappin-Scott, H. M. (1997). Consensus model of biofilm structure.Cardiff, U.K, 1–9.

Stoodley, P., Cargo, R., Rupp, C. J, Wilson, S. and Klapper, I. (2002). Biofilm material properties as related to shear-induced deformation and detachment phenomena.Journal of Industrial Microbiology and Biotechnology29, 361-367.

Xiong, P., Jia, Z., Zhou, W., Yan, J., Wang, P., Yuan, W., Li, Y., Cheng, Y., Guan, Z. and Zheng, Y. (2019). Osteogenic and pH stimuli-responsive self-healing coating on biomedical Mg-1Ca alloy, Acta Biomaterialia92, 336−350.

How to Cite
Baishya, R., Chatterjee, R., Banerjee, S., & Hasnain, M. (2020). Application of FESEM and FTIR for evaluation of Staphylococcus aureus biofilms grown on chitin and polycarbonate membrane. Revista Mexicana De Ingeniería Química, 20(1), 509-519.

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