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

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.