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Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish


Mycobacterium marinum–infected zebrafish are used to study tuberculosis pathogenesis, as well as for antitubercular drug discovery. The small size of zebrafish larvae coupled with their optical transparency allows for rapid analysis of bacterial burdens and host survival in response to genetic and pharmacological manipulations of both mycobacteria and host. Automated fluorescence microscopy and automated plate fluorimetry (APF) are coupled with facile husbandry to facilitate large-scale, repeated analysis of individual infected fish. Both methods allow for in vivo screening of chemical libraries, requiring only 0.1 μmol of drug per fish to assess efficacy; they also permit a more detailed evaluation of the individual stages of tuberculosis pathogenesis. Here we describe a 16-h protocol spanning 22 d, in which zebrafish larvae are infected via the two primary injection sites, the hindbrain ventricle and caudal vein; this is followed by the high-throughput evaluation of pathogenesis and antimicrobial efficacy.

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Figure 1: Single-cell preparation.
Figure 2: Microinjection sites.
Figure 3: VAMP.
Figure 4: Experimental results.

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We thank F. Roca for images of intracellular bacteria and larval illustrations; C.J. Cambier for macrophage recruitment images and movies; and C. Cosma, M. Troll, D. Berry, D. Tobin, J. Cameron and R. Berg for helpful discussion and protocol development.

Author information

Authors and Affiliations



J.M.D. developed the caudal vein injection technique and macrophage recruitment assay. K.T. conceived and developed the single-cell protocol, VAMP, cryo-anesthesia, high-throughput microscopy and the autofluorescence-based survival assay. K.T. developed the fluorescence constructs and APF. K.T. performed the experiments and tutorial videos. K.W. developed FPC. L.R. conceived the larval infection model and FPC and guided the development of all protocols. K.T. and L.R. wrote the manuscript.

Corresponding author

Correspondence to Lalita Ramakrishnan.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

General setup of microinjection station and micromanipulator. (a) Safe and efficient injection of larval zebrafish is achieved with a well-designed injection station. (b) The micromanipulator should be setup at right angles, with each knob giving exclusive control to x, y, and z-planes, and with the needle angled downwards to approximately 30°. After the initial setup, and during injections, the micromanipulator will only be moved in the z-plane by control of the z-knob (red arrow). (PDF 2508 kb)

Supplementary Figure 2

Microinjection Metrics (a) High resolution image of microinjection needle. Outer diameter of needle tip is approximately 10 μm. Note that the point of the needle may be beveled, blunt or irregular. Although some users prefer one type over another, microinjection can be performed with all types of needle points. Scale bar 50 μm. (b) Microinjection into mineral oil corresponding with caudal vein and hindbrain microinjection volumes. Microinjection diameters average at 139 nm with a calculated volume of 1.4 nL. Scale bar 200 μm. (PDF 1281 kb)

Supplementary Method

ImageJ FPC Macro (TXT 0 kb)

Supplementary Video 1

Hindbrain injections (MPG 23972 kb)

Supplementary Video 2

Hindbrain injections via the forebrain (MPG 26782 kb)

Supplementary Video 3

Syringing bacteria (MPG 19126 kb)

Supplementary Video 4

Needle break (MPG 7302 kb)

Supplementary Video 5

Caudal vein injection (MPG 23086 kb)

Supplementary Video 6

Macrophage recruitment (MOV 64864 kb)

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Takaki, K., Davis, J., Winglee, K. et al. Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish. Nat Protoc 8, 1114–1124 (2013).

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