Hostname: page-component-76fb5796d-vfjqv Total loading time: 0 Render date: 2024-04-26T15:01:05.126Z Has data issue: false hasContentIssue false
  • English
  • Français

Extracellular Matrix Reorganization during Cryo Preparation for Scanning Electron Microscope Imaging of Staphylococcus aureus Biofilms

Published online by Cambridge University Press:  04 July 2014

Yong Wu ,
Jing Liang ,
Kim Rensing ,
Tseng-Ming Chou  and
Matthew Libera
Yong Wu
Affiliation:
Department of Chemical Engineering and Materials Science, Stevens Institute of Technology, Hoboken, NJ 07030, USA
Jing Liang
Affiliation:
Department of Chemical Engineering and Materials Science, Stevens Institute of Technology, Hoboken, NJ 07030, USA
Kim Rensing
Affiliation:
Leica Microsystems Inc., 1700 Leider Lane, Buffalo Grove, IL 60089, USA
Tseng-Ming Chou
Affiliation:
Department of Chemical Engineering and Materials Science, Stevens Institute of Technology, Hoboken, NJ 07030, USA
Matthew Libera*
Affiliation:
Department of Chemical Engineering and Materials Science, Stevens Institute of Technology, Hoboken, NJ 07030, USA
*
*Corresponding author. mlibera@stevens.edu
Get access

Abstract

Biofilms are three-dimensional communities of bacteria distributed in a highly hydrated extracellular matrix (ECM). They can be visualized by scanning electron microscopy (SEM), but the requisite SEM sample preparation can modify the biofilm morphology. Here, four different approaches to prepare biofilms of hydrated Staphylococcus aureus for SEM imaging are compared. In order of increasing cooling effectiveness these are: (1) drying in air; (2) plunging in liquid nitrogen; (3) plunging in liquid ethane; and (4) high pressure freezing with liquid nitrogen. These different methods give rise to markedly different biofilm morphologies, which are revealed by cryo-SEM imaging. Significantly, high-pressure frozen biofilms exhibit a rich network of nanoscale ECM fibers surrounding individual bacteria throughout the biofilm thickness. This structure is entirely lost when similar biofilms are dried in air, and it is substantially modified when these biofilms are plunged into liquid nitrogen or liquid ethane.

Keywords

biofilmextracellular matrixscanning electron microscopycryoS. aureus
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 5 , October 2014 , pp. 1348 - 1355
Copyright
© Microscopy Society of America 2014 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Al-Amoudi, A., Chang, J.J., Leforestier, A., McDowall, A., Salamin, L.M., Norlen, L.P., Richter, K., Blanc, N.S., Studer, D. & Dubochet, J. (2004). Cryo-electron microscopy of vitreous sections. EMBO J 23(18), 3583–3588.CrossRefGoogle ScholarPubMed
Alhede, M., Qvortrup, K., Liebrechts, R., Hoiby, N., Givskov, M. & Bjarnsholt, T. (2012). Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition. FEMS Immunol Med Microbiol 65(2), 335–342.CrossRefGoogle ScholarPubMed
Barnes, A.M., Ballering, K.S., Leibman, R.S., Wells, C.L. & Dunny, G.M. (2012). Enterococcus faecalis produces abundant extracellular structures containing DNA in the absence of cell lysis during early biofilm formation. MBio 3(4), e00193–e00202.CrossRefGoogle ScholarPubMed
Bleck, C.K., Merz, A., Gutierrez, M.G., Walther, P., Dubochet, J., Zuber, B. & Griffiths, G. (2010). Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis. J Microsc 237(1), 23–38.CrossRefGoogle ScholarPubMed
Bontognali, T.R.R., Vasconcelos, C., Warthmann, R.J., Dupraz, C., Bernasconi, S.M. & McKenzie, J.A. (2008). Microbes produce nanobacteria-like structures, avoiding cell entombment. Geology 36(8), 663–666.CrossRefGoogle Scholar
Costerton, J.W., Stewart, P.S. & Greenberg, E.P. (1999). Bacterial biofilms: a common cause of persistent infections. Science 284(5418), 1318–1322.CrossRefGoogle ScholarPubMed
Dubochet, J., Adrian, M., Chang, J., Homo, J., Lepault, J., McDowall, A.W. & Schultz, P. (1988). Cryo-electron microscopy of vitrified specimens. Q Rev Biophys 21, 129–228.CrossRefGoogle ScholarPubMed
Dubochet, J., Chang, J.-J., Freeman, R., Lepault, J. & McDowall, A.W. (1982). Frozen aqueous suspensions. Ultramicroscopy 10, 55–62.CrossRefGoogle Scholar
Erlandsen, S., Lei, M., Martin-Lacave, I., Dunny, G. & Wells, C. (2003). High resolution CryoFESEM of microbial surfaces. Microsc Microanal 9(4), 273–278.CrossRefGoogle ScholarPubMed
Flemming, H.C., Meier, M. & Schild, T. (2013). Mini-review: Microbial problems in paper production. Biofouling 29(6), 683–696.CrossRefGoogle ScholarPubMed
Flemming, H.C. & Wingender, J. (2010). The biofilm matrix. Nat Rev Microbiol 8(9), 623–633.CrossRefGoogle ScholarPubMed
Gomes, F., Teixeira, P., Cerca, N., Azeredo, J. & Oliveira, R. (2011). Effect of farnesol on structure and composition of Staphylococcus epidermidis biofilm matrix. Curr Microbiol 63(4), 354–359.CrossRefGoogle ScholarPubMed
Harris, L.G. & Richards, R.G. (2004). Staphylococcus aureus adhesion to different treated titanium surfaces. J Mater Sci Mater Med 15(4), 311–314.CrossRefGoogle ScholarPubMed
Harris, L.G. & Richards, R.G. (2006). Staphylococci and implant surfaces: A review. Injury 37(Suppl 2), S3–S14.CrossRefGoogle ScholarPubMed
Hwang, G., Kang, S., El-Din, M.G. & Liu, Y. (2012). Impact of an extracellular polymeric substance (EPS) precoating on the initial adhesion of Burkholderia cepacia and Pseudomonas aeruginosa. Biofouling 28(6), 525–538.CrossRefGoogle ScholarPubMed
Kanno, H., Speedy, R.J. & Angell, C.A. (1975). Supercooling of water to −92°C under pressure. Science 189(4206), 880–881.CrossRefGoogle Scholar
Kensche, A., Basche, S., Bowen, W.H., Hannig, M. & Hannig, C. (2013). Fluorescence microscopic visualization of non cellular components during initial bioadhesion in situ. Arch Oral Biol 58(10), 1271–1281.CrossRefGoogle ScholarPubMed
Langer, J.S. (1980). Instabilities and pattern formation in crystal growth. Rev Mod Phys 52(1), 1–30.CrossRefGoogle Scholar
Melo, L.F. (2005). Biofilm physical structure, internal diffusivity and tortuosity. Water Sci Technol 52(7), 77–84.CrossRefGoogle Scholar
Pitt, W.G., McBride, M.O., Barton, A.J. & Sagers, R.D. (1993). Air-water interface displaces adsorbed bacteria. Biomaterials 14(8), 605–608.CrossRefGoogle ScholarPubMed
Qin, Z., Ou, Y., Yang, L., Zhu, Y., Tolker-Nielsen, T., Molin, S. & Qu, D. (2007). Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis. Microbiology 153(Pt 7), 2083–2092.CrossRefGoogle ScholarPubMed
Studer, D., Muller, M. & Michel, M. (1989). High pressure freezing comes of age. Scanning Microsc 3(Suppl 3), 253–269.Google ScholarPubMed
Vanhecke, D., Graber, W. & Studer, D. (2008). Close-to-native ultrastructural preservation by high pressure freezing. In Methods in Cell Biology, Allen T.D. (Ed.), pp. 151–164. San Diego, California: Academic Press.Google Scholar
Wingender, J., Strathmann, M., Rode, A., Leis, A. & Flemming, H.C. (2001). Isolation and biochemical characterization of extracellular polymeric substances from Pseudomonas aeruginosa. Methods Enzymol 336, 302–314.CrossRefGoogle ScholarPubMed