Source: Anderson, L. S., et al. A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection. J. Vis. Exp. (2019).
This video demonstrates a technique to generate a murine model of skin wound infection. Upon infecting mice — engineered to express a fluorescent protein in neutrophils — with a genetically modified bioluminescent Staphylococcus aureus — a pathogenic bacteria. The bacterial burden at the infection site and the neutrophil recruitment dynamics can be studied using whole animal imaging.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mouse Source and Housing
2. Bacterial Preparation and Quantification
3. Excisional Skin Wounding and Inoculation with S. aureus
4. S. aureus Inoculation
5. In Vivo bioluminescence imaging (BLI) and fluorescence imaging (FLI)
Figure 1: Correlation between OD600 and CFU counts for ALC2906. 3-4 ALC2906 SH1000 colonies were picked from an agar plate and transferred into TSB with 10 µg/mL chloramphenicol for overnight culture. The next day, the suspension was split 1:50 into TSB with 10 µg/mL chloramphenicol and cultured. Optical density at 600 nm (OD600) was measured in regular intervals after 2 hours using a spectrophotometer. At each measurement, the bacteria were diluted 1:100,000 in ice-cold PBS and aliquoted onto an agar plate for overnight incubation. CFUs were counted the following day to calculate the initial concentration and correlated to OD600. N = 3 with 4 different OD600 measurements per experiment.
The authors have nothing to disclose.
14 mL Polypropylene Round-Bottom Tube | Falcon | 352059 | |
6mm Disposable Biopsy Punch | Integra Miltex | 33-36 | |
Bioluminescent S. aureus | Lloyd Miller, Johns Hopkins | ALC 2906 SH1000 | |
Bovine Blood Agar, 5%, Hardy Diagnostics | VWR | 10118-938 | |
Buprenoprhine hydrochloride injectable | Western Medical Supply | 7292 | 0.3 mg/mL |
C57BL/6J Mice | Jackson Labratory | 664 | |
Chloramphenicol (crystalline powder) | Fisher BioReagents | BP904-100 | |
DPBS (1X) | Gibco | 14190-144 | |
Insulin Syringes | Becton, Dickson and Company | 329461 | .35 mm (28 G) x 12.7 mm (1/2'') |
IVIS Spectrum In Vivo Imaging System | Perkin Elmer | 124262 | |
Living Image Software – IVIS Spectrum Series | Perkin Elmer | 128113 | |
LysM-eGFP Mice | Thomas Graff Albert Einstein College of New York | NA | |
Microvolume Spectrophotometer | ThermoFisher Scientific | ND-2000 | |
MyD88 KO Mice | Jackson Labratory | 9088 | |
Non-woven sponges | AMD- Ritmed Inc | A2101-CH | 5 cm x 5 cm |
Povidone Iodine 10% Solution | Aplicare | 697731 | |
Tryptic Soy Broth | Becton, Dickson and Company | 211825 |