Skip to main content
Log in

Detecting Salmonella Type II flagella production by transmission electron microscopy and immunocytochemistry

  • Protocol
  • Published:
Journal of Microbiology Aims and scope Submit manuscript

Abstract

The bacterial flagellum is an appendage structure that provides a means for motility to promote survival in fluctuating environments. For the intracellular pathogen Salmonella enterica serovar Typhimurium to survive within macrophages, flagellar gene expression must be tightly regulated, and thus, is controlled at multiple levels, including DNA recombination, transcription, post-transcription, protein synthesis, and assembly within host cells. To understand the contribution of flagella to Salmonella pathogenesis within the host, it is critical to detect flagella production within macrophages via microscopy. In this paper, we describe two methods for detecting bacterial flagella by microscopy both in vitro and in vivo infection models.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Brenner, S. and Horne, R.W. 1959. A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta34, 103–110.

    Article  CAS  Google Scholar 

  • Cattini, P.A. and Davies, H.G. 1984. Observations on the kinetics of uranyl acetate and phosphotungstic acid staining of chromatin in thin sections for electron microscopy. Stain Technol.59, 291–304.

    Article  CAS  Google Scholar 

  • Chevance, F.F. and Hughes, K.T. 2008. Coordinating assembly of a bacterial macromolecular machine. Nat. Rev. Microbiol.6, 455–465.

    Article  CAS  Google Scholar 

  • Choi, E., Han, Y., Cho, Y.J., Nam, D., and Lee, E.J. 2017. A trans-acting leader RNA from a Salmonella virulence gene. Proc. Natl. Acad. Sci. USA114, 10232–10237.

    Article  CAS  Google Scholar 

  • Datsenko, K.A. and Wanner, B.L. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA97, 6640–6645.

    Article  CAS  Google Scholar 

  • De Carlo, S. and Harris, J.R. 2011. Negative staining and cryo-negative staining of macromolecules and viruses for TEM. Micron42, 117–131.

    Article  CAS  Google Scholar 

  • de Vries, N., Zwaagstra, K.A., Huis in’t Veld, J.H., van Knapen, F., van Zijderveld, F.G., and Kusters, J.G. 1998. Production of monoclonal antibodies specific for the i and 1,2 flagellar antigens of Salmonella typhimurium and characterization of their respective epitopes. Appl. Environ. Microbiol.64, 5033–5038.

    Article  CAS  Google Scholar 

  • Drevets, D.A., Canono, B.P., Leenen, P.J., and Campbell, P.A. 1994. Gentamicin kills intracellular Listeria monocytogenes. Infect. Immun.62, 2222–2228.

    Article  CAS  Google Scholar 

  • Eriksson, S., Lucchini, S., Thompson, A., Rhen, M., and Hinton, J.C. 2003. Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol. Microbiol.47, 103–118.

    Article  CAS  Google Scholar 

  • Garcia-del Portillo, F. 2001. Salmonella intracellular proliferation: where, when and how? Microbes Infect.3, 1305–1311.

    Article  CAS  Google Scholar 

  • Griffiths, G. 1993. Introduction to immunocytochemistry and historical background. In Fine structure immunocytochemistry, pp. 1–8. Springer Berlin Heidelberg, Berlin, Heidelberg, Germany.

    Chapter  Google Scholar 

  • Hautefort, I., Thompson, A., Eriksson-Ygberg, S., Parker, M.L., Lucchini, S., Danino, V., Bongaerts, R.J., Ahmad, N., Rhen, M., and Hinton, J.C. 2008. During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems. Cell. Microbiol.10, 958–984.

    Article  CAS  Google Scholar 

  • Hayat, M.A. 2000 Principles and techniques of electron microscopy: biological applications. Cambridge University Press, Cambridge, UK; New York.

    Google Scholar 

  • Ikeda, J.S., Schmitt, C.K., Darnell, S.C., Watson, P.R., Bispham, J., Wallis, T.S., Weinstein, D.L., Metcalf, E.S., Adams, P., O’Connor, C.D., et al. 2001. Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis. Infect. Immun.69, 3021–3030.

    Article  CAS  Google Scholar 

  • Kim, K.W., Lee, I.J., Hyun, J.W., Lee, Y.H., and Park, E.W. 2010. Different profiles of the negatively stained citrus canker bacterium Xanthomonas citri pv. citri depending on culture media and heavy metal stains. Plant Pathol. J.26, 90–92.

    Article  Google Scholar 

  • Knodler, L.A., Vallance, B.A., Celli, J., Winfree, S., Hansen, B., Montero, M., and Steele-Mortimer, O. 2010. Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. Proc. Natl. Acad. Sci. USA107, 17733–17738.

    Article  CAS  Google Scholar 

  • Maloy, S.R. and Nunn, W.D. 1981. Selection for loss of tetracycline resistance by Escherichia coli. J. Bacteriol.145, 1110–1111.

    Article  CAS  Google Scholar 

  • Mandell, G.L. 1973. Interaction of intraleukocytic bacteria and antibiotics. J. Clin. Invest.52, 1673–1679.

    Article  CAS  Google Scholar 

  • Nanassy, O.Z. and Hughes, K.T. 1998. In vivo identification of intermediate stages of the DNA inversion reaction catalyzed by the Salmonella Hin recombinase. Genetics149, 1649–1663.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ohi, M., Li, Y., Cheng, Y., and Walz, T. 2004. Negative staining and image classification — Powerful tools in modern electron microscopy. Biol. Proced Online6, 23–34.

    Article  CAS  Google Scholar 

  • Renault, T.T., Abraham, A.O., Bergmiller, T., Paradis, G., Rainville, S., Charpentier, E., Guet, C.C., Tu, Y., Namba, K., Keener, J.P., et al. 2017. Bacterial flagella grow through an injection-diffusion mechanism. eLife6, pii: e23136.

    Article  Google Scholar 

  • Robin Harris, J. and Horne, R.W. 1994. Negative staining: A brief assessment of current technical benefits, limitations and future possibilities. Micron25, 5–13.

    Article  Google Scholar 

  • Samatey, F.A., Imada, K., Nagashima, S., Vonderviszt, F., Kumasaka, T., Yamamoto, M., and Namba, K. 2001. Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature410, 331–337.

    Article  CAS  Google Scholar 

  • Sano, G., Takada, Y., Goto, S., Maruyama, K., Shindo, Y., Oka, K., Matsui, H., and Matsuo, K. 2007. Flagella facilitate escape of Salmonella from oncotic macrophages. J. Bacteriol.189, 8224–8232.

    Article  CAS  Google Scholar 

  • Schmitt, C.K., Ikeda, J.S., Darnell, S.C., Watson, P.R., Bispham, J., Wallis, T.S., Weinstein, D.L., Metcalf, E.S., and O’Brien, A.D. 2001. Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis. Infect. Immun.69, 5619–5625.

    Article  CAS  Google Scholar 

  • Valdivia, R.H. and Falkow, S. 1996. Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol. Microbiol.22, 367–378.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2019R1A2C2003460) and by grants from Korea University (K1823071 and K1821661) to EL.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Eun-Jin Lee.

Ethics declarations

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Han, Y., Lee, EJ. Detecting Salmonella Type II flagella production by transmission electron microscopy and immunocytochemistry. J Microbiol. 58, 245–251 (2020). https://doi.org/10.1007/s12275-020-9297-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12275-020-9297-y

Keywords

Navigation