Swab-based Conjunctival Commensal Bacteria Isolation: A Technique to Isolate Commensal Bacteria from Conjunctiva of Murine Model

Conjunctiva of the eye harbors commensal bacteria. This indigenous microflora protects the eye from infection by activating the immune cells to attack any harmful microbes that enter the eye linings.

To isolate commensal bacteria, place an anesthetized mouse in a lateral position. Take a sterile eye swab dipped in a suitable medium. Insert the wet swab tip into the medial conjunctival region of the left eye. Depress the eyeball and move the swab back and forth along the conjunctiva to collect a diverse commensal population.

Remove the swab and stir it into a tube containing microbial enrichment media. This step releases the entrapped bacteria from the cotton mesh of the swab into the media. Discard the swab and incubate the tube to facilitate the onset of bacterial growth.

Dispense a few drops of the eye swab inoculated enriched media onto a blood agar plate in the desired pattern and incubate. The casein and soy peptones present in the media supply nitrogen required for peptide synthesis while the sodium chloride maintains osmotic equilibrium.

Commensal bacteria possess hemolysin enzymes that digest the hemoglobin present in blood media. This produces a characteristic pattern on the agar plate, causing different bacteria to proliferate into morphologically distinct colonies. Count the number of colonies to determine their relative abundance in the conjunctiva.

Make sure that the mouse is properly anesthetized by squeezing a hind foot pad. Only proceed if there is no movement. Assign one hand to handle anesthetized mice and the other hand to handle the eye swab and the culture. Remove the mouse from the cage and place it on top of the work surface positioned on its side with the left eye exposed. Spray gloved hands with isopropanol and dry them with a paper towel.

Uncap a labeled BHI microcentrifuge tube with the dedicated media handling hand and place the tube back in the rack. Dip the cotton-coated tip of the eye swab in the BHI. Then, withdraw the swab from the tube while swirling the tip twice against the inner tube to remove excess liquid and remove it.

With the mouse handling hand, gently hold the mouse by the scruff of the neck. With the other hand, place the tip of the eye swab against the medial conjunctival region of the left eye. Lightly depress the eyeball and move the swab in a window washing motion between the lower eyelid and eye 10 times, maintaining constant pressure. Without touching the fur, gently remove the tip of the swab perpendicular to where it was inserted. Place the swab cotton side down directly into a labeled microcentrifuge tube with BHI media.

Apply an eye drop to the swabbed eye. If desired, acquire skin or fur swabs for control samples, sterilizing gloves appropriately between each swab. When finished, return the mouse to the cage. Let the swab stand for 10 to 15 minutes on ice. Then, sterilize gloved hands and remove the swab while mixing the tip in the media for 10 rotations. Withdraw the swab by swirling the tip against the inner wall of the tube for five rotations and dispose of it in a biohazard container. Repeat the process for each mouse.

Enrich the sample by incubating the tubes statically for 1 hour at 37 degrees Celsius. During incubation, label one room temperature TSA plate per mouse eye swab or control swab and divide it in half. Remove the enriched samples from the incubator and place them on ice. Briefly vortex the samples to mix. Then, aliquot 10 microliters of the sample onto the TSA plate and tilt the plate to form a strip. Repeat this twice.

On the other side of the plate's dividing line, create 10 dots with 10 microliters of sample each. Incubate the plates at 37 degrees Celsius for 18 hours, 2 days, and 4 days in a clean chamber that prevents agar plates from drying. Count the colonies in the strips. Note morphology and calculate colony forming units per swab for morphologically similar isolates.