Source: Thrikawala, S., et al. Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions. J. Vis. Exp. (2020).
This video demonstrates a method to quantify fungal infection in zebrafish larvae. Fungal spores are microinjected into the larval hindbrain ventricle, and the larvae are incubated to allow infection development. Infected larvae are harvested at defined intervals, and infection progress is assessed by enumerating colony-forming units.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Aspergillus spores for injection
2. Preparation of agar plates for injection
3. Zebrafish larva hindbrain ventricle microinjection
4. Establishment of injected and viable spore numbers
The authors have nothing to disclose.
Dumont forceps #5 | Roboz Surgical Instrument Co. | RS-5045 | |
Eyepiece reticle | Microscope World | RETR10 | For calibrating needles, used in Stereomicroscope |
Microinjector setup: Back pressure unit | Applied Scientific Instrumentation | BPU | |
Footswitch | Applied Scientific Instrumentation | FTSW | |
Micro pipet holder kit | Applied Scientific Instrumentation | M-Pip | |
Pressure injector | Applied Scientific Instrumentation | MPPI-3 | |
Micromanipulator setup: Micromanipulator | Narashige (Tritech) | M-152 | |
Magnetic stand and plate | Tritech | MINJ-HBMB | |
Needle puller | Sutter Instrument | P-97 | |
Stereomicroscope | Nikon | SMZ-745 | |
Tissuelyser II | Qiagen | 85300 | To homogenize larvae |
Material | |||
Agarose | Fisher | BP160-500 | |
Ampicillin sodium salt | Fisher | AAJ6380706 | |
BSA, fraction V | VWR | AAJ65855-22 | |
Kanamycin sulfate | Fisher | AAJ1792406 | |
L spreaders | Fisher | 14 665 230 | |
Microcapillary needles (no filament) | World Precision Instruments (WPI) | TW100-3 | |
Microloader pipet tips | VWR | 89009-310 | To load the needle with Aspergillus suspension |
Miracloth | VWR | EM475855-1R | To filter Aspergillus suspension |
N-phenylthiourea | Fisher | AAL0669009 | To prevent pigmentation |
Phenol red, 1% solution | Fisher | 57254 | |
Tricaine (Ethyl 3-aminobenzoate, methanesulfonic acid salt) | Fisher | AC118000500 | To anesthetize larvae |
Tween-20 | Fisher | BP337-500 | |
Media and Solutions | Components/Recipe | ||
E3 media: 60x E3 | 17.2 g NaCl, 0.76 g KCl, 2.9 g CaCl2, 4.9 g MgSO4 · 7H2O, to 1 L with H2O | ||
1x E3 | 16.7 ml 60x stock, 430 ul 0.05 M NaOH, to 1 L with H2O (optional: + 3 ml 0.01% methylene blue) | ||
Tricaine stock solution | 2 g Tricaine, 5 g Na2HPO4 · 7H2O, 4.2 ml 60X E3, to 500 ml with H2O, pH to 7.0-7.5 with NaOH | ||
Glucose minimal media (GMM) agar: GMM agar | 10 g Glucose (Dextrose), 50 ml 20x Nitrate salts, 1 ml TE, to 1 L with H2O, pH to 6.5 with NaOH, + 16 g Agar, autoclave | ||
20x Nitrate salts | 120 g NaNO3, 10.4 g KCl, 10.4 g, MgSO4 · 7H2O, 30.4 g, KH2PO4, to 1 L with H2O, autoclave | ||
Trace elements (TE) | 2.20 g ZnSO4 · 7H2O, 1.10 g H3BO3, 0.50 g MnCl2 · 4H2O, 0.16 g FeSO4 · 7H2O, 0.16 g CoCl2 · 6H2O, 0.16 g CuSO4 · 5H2O, 0.11 g (NH4)6Mo7O24 · 4H2O, 5.00 g Na2EDTA, to 100 ml with H2O, dissolve stirring overnight, autoclave |