Quantification of Bacterial Load in Mouse Spleen and Liver After Systemic Infection

תקציר

Source: Ahn, J. J., et al. Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses. J. Vis. Exp. (2016)

The video demonstrates bacterial load measurement in spleen and liver tissues isolated from mice infected with an intracellular bacterial pathogen. Bacteria, accumulated within tissue-resident phagocytes during systemic infection, are released using detergent-based lysis and mechanical disruption. Serial dilutions of the homogenates are plated on nutrient agar, and following incubation, colony counts indicate the bacterial burden within each organ.

פרוטוקול

Disclaimer Text: All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Glycerol Stocks for Long-term Storage

NOTE: This procedure describes how glycerol stocks of the EGD strain of L. monocytogenes are prepared from an original glycerol stock. Steps that have the potential to generate aerosols should be performed within a certified biosafety cabinet (BSC).

1. Prepare brain heart infusion (BHI) agar plates for bacterial growth. For this, add 3.8% (w/v) BHI broth and 1.5% (w/v) agar to double-distilled wate....

חומרים

רשימת החומרים שנעשה בהם שימוש במאמר זה

שם	חברה	מספר קטלוג	הערות
Brain Heart Infusion Broth, Modified	BD	299070	any brand should be appropriate
Agar	BD	214010	any brand should be appropriate
Triton X-100	Sigma-Aldrich	X100	any brand should be appropriate
1x PBS	Sigma	D8537	any brand should be appropriate
TissueLyser II	Qiagen	85300	any brand should be appropriate
16% Paraformaldehye	Electron Microscopy Sciences	15710	Dilute to 4% PFA in ddH2O or 1x PBS
10x Perm/Wash buffer	BD	554723	Dilute 1x in ddH2O
OneComp eBeads	eBioscience	01-1111	Use one drop per stain
50 ml vented tubes for culture			Used for culturing the bacteria, any brand should be appropriate
1.5 ml microcentrifuge tubes			any brand should be appropriate
Bacterial petri dishes			any brand should be appropriate
2 ml cyrovials			any brand should be appropriate
UV spectrometer			any brand should be appropriate
safety engineered needles			any brand should be appropriate
C57BL/6J	Jackson laboratories	Stock#000664	Order for arrival at 7 weeks
70% Ethanol			For decontamination
Glass beads			any brand should be appropriate
Centrifuge			rotor, buckets, bucket covers.
Microcentrifuge			any brand should be appropriate
Sterile Glycerol			any brand should be appropriate
Pipette Tips			any brand should be appropriate
Pipette			any brand should be appropriate
Surgical instruments			any brand should be appropriate
3 ml syringe			any brand should be appropriate
Filtration Units			any brand should be appropriate
Microbank Bacterial Preservation System	Pro-lab Diagnostics		Used as an alternative to glycerol stocks for long-term storage of bacteria