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

Overview

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 inhibitor into each autoclaved harvest tube. Label the tubes and place on ice. (Figure 1A).

2. Harvesting the eyes

1. Perform all procedures in this section under Biosafety Level 2 conditions.
2. Euthanize the mouse by CO₂ inhalation. Use a secondary method to confirm euthanasia.
3. Hold the euthanized mouse head secure and open the fine tip forceps on either side of the infected eye. Push down towards the head to proptose the eye. Once the tongs are behind the globe of the eye, squeeze the tongs together. Pull forceps away from the head to detach the eyeball (Figure 1B).
4. Immediately place the eyeball into a labeled harvesting tube. Place tubes on ice for no more than 60 min (Figure 1C).

3. Intraocular bacterial count

1. Perform all procedures in this section under Biosafety Level 2 conditions.
2. Confirm that all harvest tubes are tightly closed and are balanced while in the tissue homogenizer (Figure 2A). Turn on the tissue homogenizer for 1 min to homogenize the samples. Wait for 30 s, then turn on for another minute. Place tubes on ice (Figure 2B,C).
3. Serially dilute each sample 10-fold by sequentially transferring 20 μL of the homogenate into 180 μL of sterile 1x PBS. Dilute until a factor of 10^-7 is reached (Figure 2D).
4. Label each row of a warm, square BHI plate with the proper dilution factors. Transfer 10 μL of each dilution in a row to the top BHI plate that is tilted approximately 45°. Let the sample run until it almost reaches the bottom of the plate, then lay the plate flat (Figure 2E).
5. When sample is absorbed into the BHI agar, transfer the plate to a 37 °C incubator. Colonies should begin to be visible 8 h after being placed in the incubator.
6. Remove the plate from the incubator before the growth of the B. cereus colonies interferes with identifying individual colonies (Figure 2F).
7. For an accurate representation of the concentration in the sample, count the row that has between 10-100 colonies. For example, a row with the dilution fraction of 10^-4 that has 45 colonies will have a concentration of 4.5 x 10⁵ CFU/mL.
8. To calculate the total number of bacteria per eye, multiply the concentration by 0.4, which represents the millilitre volume of 1x PBS used to homogenize the eye. For example, the 4.5 x 10⁵ CFU/mL concentration would translate to 1.8 x 10⁵ CFU B. cereus per eye.

Representative 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 of the infected eye. (C) Harvest tube containing a mouse eye.

Figure 2: Processing the harvested eye for intraocular bacterial count. (A) Harvest tube with infected eye clamped tightly to a tissue homogenizer. (B) Infected eyes were homogenized twice for 1 min each. Track dilution (D) of the eye homogenates (C) and subsequent plating (E) for the bacterial quantitation. (E) Representative individual Bacillus cereus colony after track dilution.

Materials

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