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Methods for generating and colonizing gnotobiotic zebrafish

Nature Protocols volume 3, pages 1862–1875 (2008)Cite this article

Abstract

Vertebrates are colonized at birth by complex and dynamic communities of microorganisms that can contribute significantly to host health and disease. The ability to raise animals in the absence of microorganisms has been a powerful tool for elucidating the relationships between animal hosts and their microbial residents. The optical transparency of the developing zebrafish and relative ease of generating germ-free (GF) zebrafish make it an attractive model organism for gnotobiotic research. Here we provide a protocol for generating zebrafish embryos; deriving and rearing GF zebrafish; and colonizing zebrafish with microorganisms. Using these methods, we typically obtain 80–90% sterility rates in our GF derivations with 90% survival in GF animals and 50–90% survival in colonized animals through larval stages. Obtaining embryos for derivation requires approximately 1–2 h, with a 3- to 8-h incubation period before derivation. Derivation of GF animals takes 1–1.5 h, and daily maintenance requires 1–2 h.

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Figure 1
Figure 2: Schematic diagram of zebrafish laparotomy.
Figure 3: Overview of gnotobiotic zebrafish husbandry.
Figure 4: Comparison of colonization methods in zebrafish.

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Acknowledgements

This work was supported by the National Institutes of Health (DK073695, DK34987, DK07737 and RR018603) and the University of North Carolina at Chapel Hill. We thank Maureen Bower, Roger Orcutt, Chad Trent, Frank Kanther, Erika Mittge and Karen Guillemin for helpful discussions and protocol development, and Jon Gerler for assistance with illustrations.

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Authors and Affiliations

  1. Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Linh N Pham, Michelle Kanther, Ivana Semova & John F Rawls

  2. Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Michelle Kanther

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  1. Linh N Pham
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Correspondence to John F Rawls.

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Pham, L., Kanther, M., Semova, I. et al. Methods for generating and colonizing gnotobiotic zebrafish. Nat Protoc 3, 1862–1875 (2008). https://doi.org/10.1038/nprot.2008.186

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