Best practices for germ-free derivation and gnotobiotic zebrafish husbandry

Abstract

All animals are ecosystems with resident microbial communities, referred to as microbiota, which play profound roles in host development, physiology, and evolution. Enabled by new DNA sequencing technologies, there is a burgeoning interest in animal-microbiota interactions, but dissecting the specific impacts of microbes on their hosts is experimentally challenging. Gnotobiology, the study of biological systems in which all members are known, enables precise experimental analysis of the necessity and sufficiency of microbes in animal biology by deriving animals germ-free (GF) and inoculating them with defined microbial lineages. Mammalian host models have long dominated gnotobiology, but we have recently adapted gnotobiotic approaches to the zebrafish (Danio rerio), an important aquatic model. Zebrafish offer several experimental attributes that enable rapid, large-scale gnotobiotic experimentation with high replication rates and exquisite optical resolution. Here we describe detailed protocols for three procedures that form the foundation of zebrafish gnotobiology: derivation of GF embryos, microbial association of GF animals, and long-term, GF husbandry. Our aim is to provide sufficient guidance in zebrafish gnotobiotic methodology to expand and enrich this exciting field of research.

Keywords: Axenic; Bacterial colonization; Germ-free; Gnotobiotic; Husbandry; Microbiome; Microbiota; Sterile; Zebrafish.

FIGURE 1

Overview of critical procedures for GF zebrafish derivation. Timeline depicts timing of procedures (see Tables 1–3 for specific details). Day refers to egg fertilization. Day −1: Adult fish are set up for breeding and tank water collected for conventionalization. Day 0: Adult zebrafish spawn and embryos are collected. If experiments demand conventional (CV) controls, embryos are segregated following collection and embryos destined for GF derivation are treated with antibiotics. Following GF derivation, embryos are placed in tissue culture flasks and conventionalized (CVZ) controls inoculated with a small amount of parental tank water collected on Day 0. Day 3: Flasks are inspected to ensure larvae have hatched; unhatched larvae are dechorionated by gently shaking the flask. Day 4: Flasks are visually inspected with phase optics to ensure sterility; Fig. 2 shows examples of contamination. Larval zebrafish can be kept in tissue culture flasks up to 8 dpf without feeding; for longer experiments axenic feeding and care should begin at 4–5 dpf.

FIGURE 2

Phase microscopy visualization of potential flask contaminants and identification of sterile debris. Microorganisms present in tank water or CVZ flasks include bacteria (A, B arrowheads), filamentous microorganisms (A, arrow) and protozoans such as amoeba (B, asterisk). Reflective, irregularly shaped debris (arrows) observed in an otherwise sterile flask (C). Scale bar (A, C) 50 μM,

FIGURE 3

Microbial association of GF zebrafish procedure overview. Schematic diagram to be used with Tables 4 and 5 Number and letter sequences match procedures in Table 5. (A) Experiments with individual bacterial isolates to relate culture density to viable CFUs/mL to enable calculation of inoculation volume; see Table 5A for sample calculations. (B) For each experiment, grow and prepare bacterial inoculum according to specifications determined in (A). Pellet and wash bacteria with sterile EM, pellet again and suspend in sterile EM. Use washed bacteria for flask inoculation. (C) (1) Prior to inoculation, inspect flask for contamination under phase optics and remove media to plate any culturable bacterial contaminants. (2) Inoculate flask with washed bacteria and mix gently. Verify density by sampling flasks following inoculation (3) and at experimental end point (4).

FIGURE 4

Long-term GF husbandry considerations. Caring for GF zebrafish on a continuum from embryonic stages to adulthood resembles the process for assembling a small fish facility. Developing housing, appropriate, stage-specific nutrition, and efficient media delivery and waste removal under sterile conditions are paramount.

FIGURE 5

Tetrahymena culture maintenance overview. Schematic diagram to be used with Tables 8–10; letters in figure correspond specifically to steps in Table 8.

FIGURE 6

Tetrahymena nutrient enhancement. (A) Tetrahymena cultured in proteose peptone medium are small and have few vacuoles. (B) Tetrahymena cultured in chicken egg yolk–enriched medium are larger and packed with large vacuoles (arrow). Scale bar, 20 μm.