Conjunctival Commensal Isolation and Identification in Mice

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

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This method identifies viable ocular conjunctival microorganisms, which is challenging due to the eyes hostile environment. It can help answer whether an ocular microbiome exists and how bacterial presence changes in eye disease. In contrast to DNA sequencing, which identifies both viable and non-viable microorganisms, this method identifies only viable microorganisms, resulting in a clearer understanding of the ocular commensal community.

Studies suggest that difficult to diagnose eye diseases such as non autoimmune and autoimmune dry eye have unique ocular bacterial presence. This method could be used to diagnose eye diseases with distinctive microbial signatures. Begin by autoclaving the appropriate amount of cotton batting and toothpicks for the number of mice to be swabbed, then pinch off 1/2 a centimeter long piece of cotton batting and tease it out by pulling on the edges to form a flat single porous layer stopping just before the batting falls apart.

Swirl the batting around one of the sharp ends of the toothpick by lightly holding the stretched out piece on the toothpick tip as it is twisted. The finished eye swab will have a very thin layer of cotton stretched over the tip extending approximately 1/2 to one centimeter away from the tip. Insert the swabs into a small beaker, swab side down then cover and autoclave them.

Clean the work area with a disinfectant to minimize contamination and aliquot 0.5 milliliters of sterile brain heart infusion, or BHI media into labeled 1.5 milliliter sterile micro centrifuge tubes. Cap the tubes in a rack and set the rack on ice. Set up workflow from left to right starting with the mouse anesthetizing station, which contains the cage with the experimental mice, empty sterile cage, room temperature anesthesia, 25 gauge needle and a one milliliter syringe.

Next, prepare an eye swabbing station that contains the aliquoted BHI on ice, sterilized eye swabs, lubricating eyedrops, biohazard container, clean paper towels, and 70%isopropanol spray. Finally, set up the plating station with room temperature blood agar plates, a 10 microliter pipette, 10 microliter sterile disposable tips, and a biohazard waste container. 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 hands 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 the labeled BHI micro centrifuge tube with the dedicated media handling hand and place the two 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 micro centrifuge 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 tube statically for one 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 plates dividing line, create 10 dots with 10 microliters of sample each. Incubate the plates at 37 degrees Celsius for 18 hours, two days and four 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. Look at the dots where unique organisms are not captured in the strips.

To facilitate the visualization, we plated bacteria densely in the middle and right plates. Typically, we observed much fewer bacteria in eye swab plates. Note that on occasion, the eye swabs may not yield recoverable bacteria.

A representative eye swab plate with morphologically diverse isolates from a C57 black six mouse is shown here. For each distinct isolate, the colonies were counted in the strip and the relative abundance was calculated and plotted. For microbiological characterization, bacteria were picked from individual mouse eye swab plates to produce a master TSA plate.

When growth appeared, additional tests were run to characterize or identify the microbes. The master plate was used to provide enough inoculum to expand the respective isolates. To identify isolates, a TSA plate was streaked and incubated overnight, and MALDI-TOF MS was performed.

Examples of isolates shown here were identified as Streptococcus acidominimus and Aerococcus viridans. Significantly different levels of commensal organisms were recovered from male and female C57 black six mice. Streptococcus acidominimus, Aerococcus viridans coagulates negative Staphylococci and E.coli were isolated from C57 black six N mice.

When attempting this protocol, keep in mind that the best outcome will be achieved by coating the swab thinly and taking time during the eye swab step. If access to MALDI-TOF MS is limited, then the isolates may be identified by microbiological and biochemical testing.

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Journal (Immunology and Infection)

Journal (Immunology and Infection)

Conjunctival Commensal Isolation and Identification in Mice

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