Mycobacterium marinum Infection Protocols

M. marinum (ATCC 927) was cultured and inoculated as described previously^{35 (link)}. However, here M. marinum was suspended in phosphate buffered saline (PBS) rather than in potassium chloride prior to infections. In the zebrafish embryos, PBS with 2% polyvinylpyrrolidone-40 and 0.3 mg/ml phenol red (Sigma-Aldrich, Missouri, USA) was used as a mycobacterial carrier solution. A volume of 1 nl was injected 0–6 hours post fertilization into the yolk sac with aluminosilicate capillary needles (Sutter instrument Co., California, USA) using a micromanipulator (Narishige International, London UK) and a PV830 Pneumatic PicoPump (World Precision Instruments, Sarasota, Florida, USA) and visualized with a Stemi 2000 microscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Survival was followed daily by inspecting the larvae under a microscope. For the adult zebrafish infections, fish were anesthetized with 0.02% 3-amino benzoic acid ethyl ester, and 5 µl of M. marinum with 0.3 mg/ml phenol red (Sigma-Aldrich, Missouri, USA) was injected into the abdominal cavity with a 30 gauge Omnican 100 insulin needle (Braun, Melsungen, Germany). The M. marinum amounts (CFU) used in both the embryonic and adult infections were verified by plating bacterial inoculates on 7H10 agar (Becton Dickinson, New Jersey, USA) plates.