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Electron Microscopy to Study the Fine Structure of the Pneumococcal Cell

  • Sven HammerschmidtEmail author
  • Manfred Rohde
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1968)

Abstract

Electron microscopy allows for studying bacterial ultrastructure at high resolutions. Two types of electron microscopes are used for this purpose. The transmission electron microscope allows for access to inner bacterial ultrastructure when imaging ultrathin sections as well as cell wall-attached structures by negative staining, whereas scanning electron microscopy allows for the detection of structures on the bacterial cell surface alone or to study the interplay between pneumococci and their host cells. This chapter deals with recommendations for well-adapted methodologies to examine pneumococcal ultrastructure in detail. Especially, we focus on the preservation of the pneumococcal capsular polysaccharide, which represents an important virulence factor of pneumococci. Since capsules are highly hydrated structures, the introduction of a new fixation protocol involving lysine acetate, ruthenium red, and osmium (LRR fixation) results in a very well-preserved capsular structure in such a way that the amount of capsular material bound on the bacterial surface can be compared within different serotypes. In our method, capsular ultrastructure is preserved without the need for serotype-specific antibodies, which have been used in other studies to preserve the pneumococcal capsule. In addition, the new LRR fixation allows for studying the presence or absence of capsular material during adhesion and invasion of pneumococci on epithelial or endothelial host cells in cell culture experiments.

Key words

Pneumococci Pneumococcal capsule Transmission electron microscopy Field emission scanning electron microscopy Cryo-FESEM LRR embedding LRWhite resin Critical point drying Infection 

References

  1. 1.
    Armbruster BL, Carlemalm E, Chiovetti R, Garavito LRM, Hobot JA, Kellenberger E, Villinger W (1982) Specimen preparation for electron microscopy using low temperature embedding resin. J Microsc 126:77–85CrossRefGoogle Scholar
  2. 2.
    Bozzola JJ, Russel LD (1999) Electron microscopy. Principles and techniques, 2nd edn. Jones and Bartlett, BostonGoogle Scholar
  3. 3.
    Carlemalm E, Garavito RM, Villiger W (1982) Resin development for electron microscopy and an analysis of embedding at low temperature. J Microsc 126:123–143CrossRefGoogle Scholar
  4. 4.
    Glauert AM, Lewis PR (2000) Biological specimen preparation for transmission electron microscopy. In: Glauert AM (ed) Practical methods in electron microscopy, vol 17. Portland Press, LondonGoogle Scholar
  5. 5.
    Graham LL, Beveridge TJ (1990) Evaluation of freeze-substitution and conventional embedding protocols for routine electron microscopic processing of eubacteria. J Bacteriol 171:2141–2149CrossRefGoogle Scholar
  6. 6.
    Hoppert M (2003) Microscopic techniques in biotechnology. Wiley-VCH, WeinheimCrossRefGoogle Scholar
  7. 7.
    Hoppert M, Holzenburg A (1998) Electron microscopy in microbiology. BIOS Scientific Publishers, OxfordGoogle Scholar
  8. 8.
    Baumeister W, Grimm R, Walz J (1999) Electron tomography of molecules and cells. Trends Cell Biol 9:81–85CrossRefGoogle Scholar
  9. 9.
    Springer EL, Roth IL (1973) The ultrastructure of the capsules of Diplococcus pneumonia and Klebsiella pneumonia stained with ruthenium red. J Gen Microbiol 74:21–31CrossRefGoogle Scholar
  10. 10.
    Geno KA, Gilbert GL, Song JY, Skovsted IC, Klugman KP, Jones C, Konradsen HB, Nahm MH (2015) Pneumococcal capsules and their types: past, present, and future. Clin Microbiol Rev 28:871–899CrossRefGoogle Scholar
  11. 11.
    Geno KA, Saad JS, Nahm MH (2017) Discovery of novel pneumococcal serotype 35D, a natural WciG-deficient variant of serotype 35B. J Clin Microbiol 55:1416–1425CrossRefGoogle Scholar
  12. 12.
    Eberhardt A, Hoyland CN, Vollmer D, Bisle S, Cleverley RM, Johnsborg O, Håvarstein LS, Lewis RJ, Vollmer W (2012) Attachment of capsular polysaccharide to the cell wall in Streptococcus pneumoniae. Microb Drug Resist 18(3):240–255CrossRefGoogle Scholar
  13. 13.
    Hammerschmidt S, Wolff S, Hocke A, Rosseau S, Müller E, Rohde M (2005) Illustration of pneumococcal polysaccharide capsule during adherence and invasion of epithelial cells. Infect Immun 73:4653–4667CrossRefGoogle Scholar
  14. 14.
    Kim JO, Weiser JN (1998) Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae. J Infect Dis 177(2):368–377CrossRefGoogle Scholar
  15. 15.
    Fassel TA, Mozdziak PE, Sanger JR, Edmiston CE Jr (1997) Paraformaldehyde effect on ruthenium red and lysine preservation and staining of the staphylococcal glycocalyx. Microsc Res Tech 36:422–427CrossRefGoogle Scholar
  16. 16.
    Fassel TA, Mozdziak PE, Sanger JR, Edmiston CE Jr (1998) Superior preservation of the staphylococcal glycocalyx with aldehyde-ruthenium red and select lysine salts using extended fixation times. Microsc Res Tech 41:291–297CrossRefGoogle Scholar
  17. 17.
    Fassel TA, Edmiston CE Jr (1999) Ruthenium red and the bacterial glycocalyx. Rev Biotechnol Histochem 74:194–212CrossRefGoogle Scholar
  18. 18.
    Luft JH (1971) Ruthenium red and violet. 1. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat Rec 171:347–368CrossRefGoogle Scholar
  19. 19.
    Luft JH (1971) Ruthenium red and violet. II. Fine structural localization in animal tissue. Anat Rec 171:369–415CrossRefGoogle Scholar
  20. 20.
    Valentine RC, Shapiro BM, Stadtman ER (1968) Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. Biochemistry 7:2143–2152CrossRefGoogle Scholar
  21. 21.
    Venable JH, Coggeshall R (1965) A simplified lead citrate stain for use in electron microscopy. J Cell Biol 25:407–408CrossRefGoogle Scholar
  22. 22.
    Bradley DE (1954) Evaporated carbon films for use in electron microscopy. Br J Appl Phys 5:65–66CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Molecular Genetics and Infection Biology, Interfaculty Institute for Genetics and Functional GenomicsUniversity of GreifswaldGreifswaldGermany
  2. 2.Central Facility for MicroscopyHZI—Helmholtz Centre for Infection ResearchBraunschweigGermany

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