Static Immersion and Injection Methods for Live Cell Imaging of Foodborne Pathogen Infections in Zebrafish Larvae

  • Macarena A. Varas
  • Javiera Ortíz-Severín
  • Andrés E. Marcoleta
  • Carlos A. Santiviago
  • Miguel L. Allende
  • Francisco P. ChávezEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1918)


Important features of host–pathogen interactions have been discovered using nonmammalian hosts. Therefore, model organisms such as the nematode Caenorhabditis elegans, the social amoeba Dictyostelium discoideum, and zebrafish ( Danio rerio ) have been increasingly used for studying bacterial pathogenesis in vivo. These host models are amenable for live cell imaging studies, which can also benefit from online resources and databases (,,, as well as from a wide repertoire of genetic and genomic tools generated over the years by the scientific community. Here, we present the protocols we developed to study bacterial dynamics within infected embryonic zebrafish. This chapter describes detailed methods to achieve infections of zebrafish larvae with the foodborne pathogen Salmonella enterica serovar Typhimurium, including embryonic zebrafish spawning and maintenance, bacterial inoculation through intravenous injections and static immersion, followed by fluorescence imaging of infected transgenic zebrafish. Methods for studying bacterial dynamics within zebrafish larvae through live cell imaging are also described.

Key words

Danio rerio Live cell imaging Static immersion Innate immunity Macrophages Neutrophils 


  1. 1.
    Festing S, Wilkinson R (2007) The ethics of animal research. Talking point on the use of animals in scientific research. EMBO Rep 8:526–530. Scholar
  2. 2.
    Rollin BE (2006) The regulation of animal research and the emergence of animal ethics: a conceptual history. Theor Med Bioeth 27:285–304. Scholar
  3. 3.
    Kurz CL, Ewbank JJ (2007) Infection in a dish: high-throughput analyses of bacterial pathogenesis. Curr Opin Microbiol 10:10–16. Scholar
  4. 4.
    Rahme LG, Stevens EJ, Wolfort SF, Shao J, Tompkins RG, Ausubel FM (1995) Common virulence factors for bacterial pathogenicity in plants and animals. Science 268:1899–1902CrossRefGoogle Scholar
  5. 5.
    Mahajan-Miklos S, Rahme LG, Ausubel FM (2000) Elucidating the molecular mechanisms of bacterial virulence using non-mammalian hosts. Mol Microbiol 37:981–988. Scholar
  6. 6.
    Kanther M, Rawls JF (2010) Host-microbe interactions in the developing zebrafish. Curr Opin Immunol 22:10–19CrossRefGoogle Scholar
  7. 7.
    Lieschke GJ, Currie PD (2007) Animal models of human disease: zebrafish swim into view. Nat Rev Genet 8:353–367. Scholar
  8. 8.
    Howe DG, Bradford YM, Conlin T, Eagle AE, Fashena D, Frazer K, Knight J, Mani P, Martin R, Moxon SAT, Paddock H, Pich C, Ramachandran S, Ruef BJ, Ruzicka L, Schaper K, Shao X, Singer A, Sprunger B, Van Slyke CE, Westerfield M (2013) ZFIN, the Zebrafish model organism database: increased support for mutants and transgenics. Nucleic Acids Res 41. Scholar
  9. 9.
    Jarvik T, Smillie C, Groisman EA, Ochman H (2010) Short-term signatures of evolutionary change in the Salmonella enterica serovar Typhimurium 14028 genome. J Bacteriol 192:560–567. Scholar
  10. 10.
    Varas M, Farina A, Diaz-Pascual F, Ortiz-Severin J, Marcoleta AE, Allende ML, Santiviago CA, Chavez FP (2017) Live-cell imaging of Salmonella Typhimurium interaction with zebrafish larvae after injection and immersion delivery methods. J Microbiol Methods 135:20–25. Scholar
  11. 11.
    Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580. Scholar
  12. 12.
    Varas M, Ortíz-Severín J, Marcoleta AE, Díaz-Pascual F, Allende ML, Santiviago CA, Chávez FP (2017) Salmonella Typhimurium induces cloacitis-like symptoms in zebrafish larvae. doi:
  13. 13.
    Renshaw S a, Loynes C a, Trushell DMI, Elworthy S, Ingham PW, Whyte MKB (2006) A transgenic zebrafish model of neutrophilic inflammation. Blood 108:3976–3978. Scholar
  14. 14.
    Helaine S, Thompson JA, Watson KG, Liu M, Boyle C, Holden DW (2010) Dynamics of intracellular bacterial replication at the single cell level. Proc Natl Acad Sci 107:3746–3751. Scholar

Copyright information

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

Authors and Affiliations

  • Macarena A. Varas
    • 1
    • 2
  • Javiera Ortíz-Severín
    • 1
  • Andrés E. Marcoleta
    • 2
  • Carlos A. Santiviago
    • 3
  • Miguel L. Allende
    • 4
  • Francisco P. Chávez
    • 1
    Email author
  1. 1.Laboratorio de Microbiología de Sistemas, Departamento de Biología, Facultad de CienciasUniversidad de ChileSantiagoChile
  2. 2.Laboratorio de Biología Estructural y Molecular, Departmento de Biología, Facultad de CienciasUniversidad de ChileSantiagoChile
  3. 3.Laboratorio de Microbiología, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y FarmacéuticasUniversidad de ChileSantiagoChile
  4. 4.Facultad de Ciencias, Centro FONDAP de Regulación del GenomaUniversidad de ChileSantiagoChile

Personalised recommendations