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

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Eye Swab Preparation, Work Field Set-up, Mouse Eye Swabbing and Sample Enrichment

1. Prepare Sterile Eye Swabs
   NOTE: Sterile eye swabs are thinly coated wooden toothpicks with a fiber thin layer of cotton batting and made in house.
   1. Autoclave appropriate amount of cotton batting and toothpicks for number of mice to be swabbed.
   2. Pinch off a half a centimeter-long piece of cotton batting with thumb and forefinger. Tease out batting by pulling on the edges with thumbs and forefingers to form a flat single porous layer, stopping just before the batting falls apart (small bits of cotton dispersed throughout the stretched batting are acceptable).
   3. 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, see Figure 1. The “completed” eye swab will have a very thin layer of cotton stretched over the tip, extending approximately one half to one centimeter away from the tip.
   4. Insert “completed” swabs into small beaker, swab side down and cover. Autoclave.
2. Set Up the Workspace
   1. Prepare anesthesia containing 2 mL of ketamine (100 mg/mL), 400 µL of xylazine (100 mg/mL) and 27.6 mL of sterile saline (0.9% NaCl in dH₂O) in a sterile 50 mL sterile centrifuge tube. Filter sterilize and aliquot anesthesia for immediate use (may be stored at 4 °C for 1 month; always allow to equilibrate to room temperature prior to use).
   2. Clear and clean the work area with disinfectant to minimize contamination.
   3. Aliquot 0.5 mL of sterile Brain Heart Infusion media (BHI) into labeled 1.5 mL sterile microcentrifuge tubes (1 tube per mouse and control); arrange in microcentrifuge rack. Set the rack on ice.
   4. Set up work flow as follows (from left to right): mouse anesthetizing station (cage containing experimental mice, empty sterile cage, room temperature anesthesia, 25 G needle and 1 mL syringe), eye swabbing station (aliquoted BHI on ice, sterilized eye swabs, clean paper towels, 70% isopropanol spray) and plating station (room temperature blood agar plates, 10 µL pipette, 10 µL sterile disposable tips, biohazard waste container).
3. Eye Swabbing
   1. Administer 10 µL of anesthesia per 1 g of mouse weight dosage of ketamine and xylazine respectively intraperitoneally to each adult mouse and place into an autoclaved cage. Test anesthetization by squeezing hindfoot pad; no movement indicates appropriate anesthetization.
   2. Assign one hand to only handle anesthetized mice and the other hand to only handle the eye swab and the culture. For swabbing, remove the fully anesthetized mouse from the cage; place on top of clean work surface positioned on its’ side with left eye exposed. Spray gloved hands with isopropanol, dry with clean paper towel.
   3. Uncap specifically labeled BHI micro-centrifuge tube with dedicated media handling hand. Place the tube back into the micro-centrifuge rack on ice.  Dip the cotton coated tip of the eye swab in BHI, withdraw the eye swab from the tube swirling the tip 2 times against the inner tube to remove excess liquid.
   4. With the mouse handling hand, gently hold the mouse by the scruff of the neck. With the other hand, place the tip of eye swab against the medial conjunctival region of the left eye, see Figure 2A. Lightly depress the eyeball and move the swab in a window washing motion (Figure 2B) back and forth, between the lower eyelid and eye, 10 times. Maintain constant pressure.
   5. Without touching the fur, gently remove the tip of swab, perpendicular to where it was inserted, and place the swab cotton side down directly into a labeled micro-centrifuge tube containing BHI media. Apply a lubricating eye drop to the swabbed eye.
   6. Return the mouse to the cage.
   7. Let the swab stand for 10 to 15 min on ice and remove eye swab with the sterilized gloved hand by mixing the tip in the media for 10 rotations. Withdraw the swab by swirling tip against the inner wall of the micro-centrifuge tube for 5 rotations. Dispose of the swab in biohazard container.
   8. Repeat steps 1.3.2 to 1.3.7 for each mouse and for control samples (skin or fur swab). Sterilize gloves appropriately between each swab.
4. Enrichment
   1. Enrich the sample by incubating the tube statically for 1 h at 37 °C
   2. During incubation, label one room temperature Trypticase Soy with 5% sheep’s blood agar plate (TSA plate) per mouse and divide in half.
   3. After eye swab culture incubation, remove enriched samples from the incubator and place on ice.
   4. Briefly vortex samples to mix and plate according to the schematic in Figure 3A. Aliquot 10 µL of the sample onto the TSA plate and tilt the plate to form a strip, repeat 2 times.
   5. On the other side of the plate’s dividing line, dot 10 µL of sample on agar, repeat 9 times.
   6. Incubate plates at 37 °C for 18 h, 2 days, and 4 days (in a clean chamber that prevents agar plates from drying). Count colonies in strips, note morphology and calculate colony forming units (CFUs) per swab for morphologically similar isolates. Look at dots for unique organisms not captured in strips.