Field Emission Scanning Electron Microscopy of Biofilm-Growing Bacteria Involved in Nosocomial Infections

  • Claudia Vuotto
  • Gianfranco DonelliEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1147)


Scanning electron microscopy (SEM) provides useful information on the shape, size, and localization within the biofilm of single bacteria as well as on the steps of biofilm formation process, on bacterial interactions, and on production of extracellular polymeric substances.

When biofilms are constituted by microbial species involved in health care-associated infections, information provided by SEM can be fruitfully used not only for basic researches but also for diagnostic purposes.

The protocols currently used in our laboratory for biofilm investigation by SEM are reported here. Particularly, the procedures to fix, dehydrate, and metalize in vitro-developed biofilms or ex vivo clinical specimens colonized by biofilm-growing microorganisms are described as well as the advantages of the observation of these samples by field emission scanning electron microscopy.

Key words

Scanning electron microscopy FESEM Biofilm ultrastructure Medical devices 



Authors are indebted with Dr. Maria Pia Balice, Director of the Clinical Chemistry and Microbiology Laboratory for kindly providing bacterial strains and explanted medical devices, and gratefully acknowledge Dr. Antonino Salvia, Director, and Dr. Angelo Rossini, Vice Director of the Medical Services of the Fondazione Santa Lucia in Rome for their advices on the clinical issues.


  1. 1.
    Giangrande A, Allaria P, Torpia R et al (1993) Ultrastructure analysis of Tenckhoff chronic peritoneal catheters used in continuous ambulatory peritoneal dialysis patients. Perit Dial Int 13:S133–S135PubMedGoogle Scholar
  2. 2.
    McLaughlin-Borlace L, Stapleton F, Matheson M, Dart JK (1998) Bacterial biofilm on contact lenses and lens storage cases in wearers with microbial keratitis. J Appl Microbiol 84:827–838PubMedCrossRefGoogle Scholar
  3. 3.
    Fux CA, Quigley M, Worel AM et al (2006) Biofilm-related infections of cerebrospinal fluid shunts. Clin Microbiol Infect 12(4):331–337PubMedCrossRefGoogle Scholar
  4. 4.
    Sangetha S, Zuraini Z, Suryani S, Sasidharan S (2009) In situ TEM and SEM studies on the antimicrobial activity and prevention of Candida albicans biofilm by Cassia spectabilis extract. Micron 40:439–443PubMedCrossRefGoogle Scholar
  5. 5.
    Hannig C, Follo M, Hellwig E, Al-Ahmad A (2010) Visualization of adherent micro-organisms using different techniques. J Med Microbiol 59:1–7PubMedCrossRefGoogle Scholar
  6. 6.
    Lattif AA, Mukherjee PK, Chandra J et al (2010) Characterization of biofilms formed by Candida parapsilosis, C. metapsilosis, and C. orthopsilosis. Int J Med Microbiol 300:265–270PubMedCrossRefGoogle Scholar
  7. 7.
    Grin I, Schwarz H, Linke D (2011) Electron microscopy techniques to study bacterial adhesion. Adv Exp Med Biol 715:257–269PubMedCrossRefGoogle Scholar
  8. 8.
    Hingston PA, Stea EC, Knøchel S, Hansen T (2013) Role of initial contamination levels, biofilm maturity and presence of salt and fat on desiccation survival of Listeria monocytogenes on stainless steel surfaces. Food Microbiol 36:46–56PubMedCrossRefGoogle Scholar
  9. 9.
    Perloff JR, Palmer JN (2005) Evidence of bacterial biofilms in a rabbit model of sinusitis. Am J Rhinol 19:1–6PubMedGoogle Scholar
  10. 10.
    Vitkov L, Hermann A, Krautgartner WD et al (2005) Chlorhexidine-induced ultrastructural alterations in oral biofilm. Microsc Res Tech 68:85–89PubMedCrossRefGoogle Scholar
  11. 11.
    Carron MA, Tran VR, Sugawa C, Coticchia JM (2006) Identification of Helicobacter pylori biofilms in human gastric mucosa. J Gastrointest Surg 10:712–717PubMedCrossRefGoogle Scholar
  12. 12.
    Passerini L, Lam K, Costerton JW, King EG (1992) Biofilms on indwelling vascular catheters. Crit Care Med 20:665–673PubMedCrossRefGoogle Scholar
  13. 13.
    Donelli G, De Paoli P, Fadda G, CVC Study Group et al (2001) A multicenter study on central venous catheter-associated infections in Italy. J Chemother 13:251–262PubMedCrossRefGoogle Scholar
  14. 14.
    Nishikawa K, Takasu A, Morita K et al (2010) Deposits on the intraluminal surface and bacterial growth in central venous catheters. J Hosp Infect 75:19–22PubMedCrossRefGoogle Scholar
  15. 15.
    Aybar Y, Ozaras R, Besirli K et al (2012) Efficacy of tigecycline and vancomycin in experimental catheter-related Staphylococcus epidermidis infection: microbiological and electron microscopic analysis of biofilm. Int J Antimicrob Agents 39:338–342PubMedCrossRefGoogle Scholar
  16. 16.
    Stickler DJ (2008) Bacterial biofilms in patients with indwelling urinary catheters. Nat Clin Pract Urol 5:598–608PubMedCrossRefGoogle Scholar
  17. 17.
    Donelli G, Guaglianone E, Di Rosa R et al (2007) Plastic biliary stent occlusion: factors involved and possible preventive approaches. Clin Med Res 5:53–60PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Guaglianone E, Cardines R, Vuotto C et al (2010) Microbial biofilms associated with biliary stent clogging. FEMS Immunol Med Microbiol 59:410–420PubMedGoogle Scholar
  19. 19.
    Bernthal NM, Stavrakis AI, Billi F et al (2010) A mouse model of post-arthroplasty Staphylo-coccus aureus joint infection to evaluate in vivo the efficacy of antimicrobial implant coatings. PLoS One 5:e12580Google Scholar
  20. 20.
    Takeuchi Y, Guggenheim B, Filieri A, Baehni P (2007) Effect of chlorhexidine/thymol and fluoride varnishes on dental biofilm formation in vitro. Eur J Oral Sci 115:468–472PubMedCrossRefGoogle Scholar
  21. 21.
    Donelli G, Francolini I, Romoli D et al (2007) Synergistic activity of dispersin B and cefamandole nafate in inhibition of staphylococcal biofilm growth on polyurethanes. Antimicrob Agents Chemother 51:2733–2740PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Ferreira JA, Carr JH, Starling CE et al (2009) Biofilm formation and effect of caspofungin on biofilm structure of Candida species bloodstream isolates. Antimicrob Agents Chemother 53:4377–4384PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Siddiqui MF, Sakinah M, Singh L, Zularisam AW (2012) Targeting N-acyl-homoserine-lactones to mitigate membrane biofouling based on quorum sensing using a biofouling reducer. J Biotechnol 161:190–197PubMedCrossRefGoogle Scholar
  24. 24.
    Wang W, Tao R, Tong Z et al (2012) Effect of a novel antimicrobial peptide chrysophsin-1 on oral pathogens and Streptococcus mutans biofilms. Peptides 33:212–219PubMedCrossRefGoogle Scholar
  25. 25.
    Donelli G, Francolini I, Ruggeri V et al (2006) Pore formers promoted release of an antifungal drug from functionalized polyurethanes to inhibit Candida colonization. J Appl Microbiol 100:615–622PubMedCrossRefGoogle Scholar
  26. 26.
    Barnes AM, Ballering KS, Leibman RS et al (2012) Enterococcus faecalis produces abundant extracellular structures containing DNA in the absence of cell lysis during early biofilm formation. MBio 3:e00193-12PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Novais A, Vuotto C, Pires J et al (2013) Diversity and biofilm-production ability among isolates of Escherichia coli phylogroup D belonging to ST69, ST393 and ST405 clonal groups. BMC Microbiol 21(13):144CrossRefGoogle Scholar
  28. 28.
    Combrouse T, Sadovskaya I, Faille C et al (2013) Quantification of the extracellular matrix of the Listeria monocytogenes biofilms of different phylogenic lineages with optimization of culture conditions. J Appl Microbiol 114:1120–1131PubMedCrossRefGoogle Scholar
  29. 29.
    Priester JH, Horst AM, Van de Werfhorst LC et al (2007) Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy. J Microbiol Methods 68:577–587PubMedCrossRefGoogle Scholar
  30. 30.
    Carr JH, Anderson RL, Favero MS (1996) Comparison of chemical dehydration and critical point drying for the stabilization and visualization of aging biofilm present on interior surfaces of PVC distribution pipe. J Appl Bacteriol 80:225–232PubMedCrossRefGoogle Scholar
  31. 31.
    Araujo JC, Téran FC, Oliveira RA et al (2003) Comparison of hexamethyldisilazane and critical point drying treatments for SEM analysis of anaerobic biofilms and granular sludge. J Electron Microsc (Tokyo) 52:429–433CrossRefGoogle Scholar
  32. 32.
    Cazaux J (2005) Recent developments and new strategies in scanning electron microscopy. J Microsc 217:16–35PubMedCrossRefGoogle Scholar
  33. 33.
    Raulio M, Wilhelmson A, Salkinoja-Salonen M, Laitila A (2009) Ultrastructure of biofilms formed on barley kernels during malting with and without starter culture. Food Microbiol 26:437–443PubMedCrossRefGoogle Scholar
  34. 34.
    Donelli G, Vuotto C, Cardines R, Mastrantonio P (2012) Biofilm-growing intestinal anaerobic bacteria. FEMS Immunol Med Microbiol 65:318–325PubMedCrossRefGoogle Scholar
  35. 35.
    Bridier A, Meylheuc T, Briandet R (2013) Realistic representation of Bacillus subtilis biofilms architecture using combined microscopy (CLSM, ESEM and FESEM). Micron 48:65–69PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Microbial Biofilm LaboratoryFondazione Santa Lucia IRCCSRomeItaly

Personalised recommendations