To initiate a Malassezia infection, confirm a lack of response to pedal reflex in an anesthetized six- to eight-week-old female C57/Black/6 mouse, and apply ointment to the animal's eyes. Use a caliper to measure the thickness of two different areas of both ears to allow calculation of the average thickness per ear. Then, apply a small piece of tape to the skin of one ear before quickly removing the tape from the ear skin five times using a new piece of tape each time.
After stripping, use a sterile pipette to apply 100 microliters of the Malassezia olive oil suspension to the dorsal side of each ear, and inject 200 microliters of sterile pre-warmed 2% glucose solution subcutaneously into the nuchal fold to support metabolism and rehydration. Then, allow the mouse to recover on a heating pad for 30 minutes with monitoring before returning the animal to its cage.
At the appropriate experimental time points, use a caliper to measure two different areas of each mouse ear to allow calculation of the average thickness per ear. To measure the fungal burden in the infected skin, add 500 microliters of sterile 0.05% NP40 and distilled water in an autoclaved 5-milliliter diameter steel ball to one 2-milliliter microcentrifuge tube per ear, and weigh each tube on a balance.
Next, harvest the ears from each animal at the base, and place the tissues into individual tubes of NP40. Weigh each tube on the balance to determine the weight of each ear sample before homogenizing the ear tissues for six minutes at 25 hertz. Then, dispense 100 microliters of each sample onto m-Dixon agar plates and distribute the suspensions homogeneously across the agar before their upside-down incubation at 30 degrees Celsius.
