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

Bacterial endophthalmitis is an inflammatory eye ailment that occurs when Bacillus cereus — a pathogenic bacteria — infects the intraocular region.

To quantify the bacterial infection, place a euthanized mouse model of bacterial endophthalmitis laterally on an operating table. Apply mild pressure under the eye to help detach the eyeball from the socket. Transfer the eyeball into a tube containing an appropriate buffer supplemented with protease inhibitor cocktail.

Homogenize the ocular tissue to disintegrate it, releasing bacteria in the suspension. The protease inhibitors present in the buffer inactivate the released cellular proteases to maintain bacterial viability. Next, serially dilute the bacterial suspension to obtain various concentrations of Bacillus cereus.

Take an angled culture plate containing the enriched agar medium and demarcate rows corresponding to each dilution concentration. Dispense a few drops from the bacterial suspension on the top of the designated row and allow it to flow down until it reaches the end of the plate, helping in the uniform spreading of bacteria. Flatten the plate and incubate.

During incubation, each bacterium uses the nutrients from agar to proliferate and form individual colonies. The number of colonies decreases, corresponding to an increase in the dilution of the bacterial suspension. Count the number of colonies in a particular row to quantify the ocular bacterial load.

At the appropriate experimental end point, add 400 microliters of PBS supplemented with protease inhibitor in one labeled autoclaved harvest tube per eye on ice, and place open, fine tip forceps on either side of the infected eye. Push the tips down towards the head to proptose the eye.

Once the tongs are behind the eye globe, squeeze the tongs together, and pull the forceps away from the head to detach the eyeball. Then, immediately place the eyeball into the appropriately labeled harvesting tube. Within 60 minutes of sample collection, place the closed harvesting tubes into a tissue homogenizer, and homogenize the samples for two one-minute homogenization periods, with a 30-second period of rest in between.

After homogenization, place the samples on ice, and use 20-microliter aliquots of homogenate to serially dilute the samples in 180 microliters of PBS per dilution until a factor of 1 times 10 to the negative 8 is reached. Next, label each row of a square pre-warmed BHI plate with the appropriate dilution concentration and with the plate tilted at a 45-degree angle, add 10 microliters of each dilution into individual rows at the top of the plate. Let each sample run until it almost reaches the bottom of the plate before laying the plate flat. When the samples have been absorbed into the agar, transfer the plate to a 37-degree Celsius incubator. Colonies should begin to be visible after eight hours of incubation.

For an accurate representation of the concentration in the sample, count the row that has between 10 to 100 colonies.