Bacterial Infection Quantitation Assay: A Method to Quantify Intraocular Bacterial Load in Mouse Model of Bacterial Endophthalmitis

Abstract

Source: Mursalin, M. H. et al., Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis. J. Vis. Exp. (2021).

This video describes the technique of quantifying bacterial infection from eye homogenates obtained from a murine model of bacterial endophthalmitis caused by Bacillus cereus. This technique helps study the host immune response and its dependency on the host and ocular bacterial gene expression in murine models.

Protocol

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

1. Harvesting tube preparation

1. Place 1 mm sterile glass beads into 1.5 mL screw cap tubes.
2. Sterilize these tubes in an autoclave on a dry setting. Let the tubes cool to room temperature before use.
3. Add 10 mL of 1x sterile phosphate-buffered saline (PBS) to a sterile 15 mL centrifuge tube.
4. Add 1 tablet of protease inhibitor cocktail tablet into the tube. Mix by vortexing.
5. Pipette 400 μL of 1x phosphate-buffered saline (PBS) containing protease....

Results

Figure 1: Harvesting mouse infected eye. Post-infection, after the desired time point, harvest infected mouse eyes using sterile tweezers. (A) PBS containing sterile glass beads. (B) Harvesting .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-20 µL pipette   	RANIN	L0696003G	NA
37oC Incubator 	Fisher Scientific	 11-690-625D	NA
Bacto Brain Heart Infusion  	BD	90003-032	NA
Cell Microinjector  	MicroData Instrument, Inc.	PM2000	NA
Fine tip forceps Thermo 	Fisher Scientific	 12-000-122	NA
Glass beads 1.0 mm  	BioSpec	11079110	NA
Incubator Shaker  	New Brunswick Scientific	NB-I2400	NA
Microcapillary Pipets 5 Microliters  	Kimble	71900-5	NA
Micro-Pipette Beveler 	 Sutter Instrument Co.	BV-10	NA
Microscope Axiostar Plus 	Zeiss		NA
Microscope OPMI Lumera 	Zeiss		NA
Mini-Beadbeater-16 	BioSpec	 Model 607	NA
Needle/Pipette Puller  	Kopf	730	NA
PBS   	GIBCO	1897315	Molecular grade
Protease Inhibitor Cocktail 	 Roche	4693159001	Molecular grade
Reverse action forceps 	 Katena 	K5-8228	NA