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.