In Vivo Analysis of Implant-Associated Bacterial Infections Using Zebrafish Embryos

Abstract

Source: Zhang, X. et. al., A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection. J. Vis. Exp. (2019)

This video demonstrates a zebrafish embryo model for studying infections linked to implanted biomaterials. Injecting Staphylococcus aureus with polystyrene microspheres enhances bacterial survival, confirming the infection-promoting role of implanted materials.

Protocol

1. Preparation of Bacteria-only and Bacteria-Microspheres Suspensions

NOTE: The Staphylococcus aureus RN4220 strain expressing mCherry fluorescent protein (S. aureus-mCherry) is used. The S. aureus RN4220 strain is mutated in the virulence regulator gene agrA (accessory gene regulator A), and, therefore, may have relatively low virulence in the zebrafish embryo model. Other S. aureus strains or other bacterial species for biomaterial-associated infection (BAI) can be used.

1. Take 4 to 5 colonies of S. aureus RN4220 bacteria from tryptic soya agar culture pla....

Results

Figure 1: Schematic procedure of co-injection of Bacteria-Microspheres suspension into the tail muscle of zebrafish embryo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tryptic soya agar	BD Difco	236950	Media preparation unit at AMC
Tryptic soya broth	BD Difco	211825
Polyvinylpyrrolidone40	Applichem	A2259.0250
10 µm diameter polystyrene microspheres (blue fluorescent)	Life technology/ThemoFisher	F8829
Glass microcapilary (1 mm O.D. x 0.78 mm I.D.)	Harvard Apparatus	30-0038
Micropipette puller instrument	Sutter Instrument Inc	Flaming p-97
Light microscope LM 20	Leica	MDG33 10450123
3-aminobenzoic acid (Tricaine)	Sigma-Aldrich	E10521-50G
Agarose MP	Roche	11388991001
Stereo fluorescent microscope LM80	Leica	MDG3610450126
Microloader pipette tips	Eppendorf	5242956.003
Micromanipulator M3301 with M10 stand	World Precision Instruments	00-42-101-0000
FemtoJet express micro-injector	Eppendorf	5248ZO100329
Microtrube 2ml pp	Sarstedt	72.693.005
Zirconia beads	Bio-connect	11079124ZX
MagNA lyser	Roche	41416401
Chloramphenicol	Sigma-Aldrich	C0378
Gyrotory shaker (for bacterial growth)	New Brunswick Scientific	G10
Zebrafish incubator	VWR	Incu-line
Cuvettes	BRAND	759015
Centrifuge	Hettich-Zentrifugen	ROTANTA 460R
Spectrometer	Pharmacia biotech	Ultrospec®2000
Forceps	Sigma-Aldrich	F6521-1EA
48 well-plates	Greiner bio-one	677180
96 well-plates	Greiner bio-one	655161
Petri-dish	Falcon	353003
Petri-dish	Biomerieux	NL-132