A Technique to Quantify Fungal Infection in Zebrafish Larvae

Abstract

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.

Protocol

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

1. From an Aspergillus spore suspension, calculate the volume needed to obtain 1 x 10⁶ spores. The volume should be 20–100 μL; if not, produce a 10x dilution in 0.01% (v/v) sterile Tween-20 (Tween-water; Table of Materials). For example, if the calculated volume is 5 µL, produce a 10x dilution and use 50 µL of the diluted solution.
   NOTE:

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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