To perform ear cytology there is no need for specialist equipment apart from cotton buds, slides, staining and a good microscope.
Otitis externa in dogs is one of the most common diseases seen in small animal practice, with a reported prevalence of 6 to 20 per cent. When otitis externa is suspected, typical diagnostic procedures include clinical evaluation, otoscopic examination and cytology of the otic exudates. Cytology of the exudates is indispensable for confirming the presence and evaluating the type of infection as it is reported to be more sensitive in the detection of microorganisms than microbial culture. The cytological findings are also invaluable in determining the treatment to use and monitoring the response to the therapy
Step 1 - If the patient allows, the ear flap should be lifted up at about 30 degrees from dorsal and the cotton bud inserted in the vertical ear canal (no need to pass it through an otoscope cone), rotated and withdrawn (Fig 1).
Step 2 - The tip of the cotton bud is rolled on to a slide. Slides with frosted ends are easier to label for identification. Cotton buds from both ears can be rolled on the same slide and the slide should be labelled to differentiate right ear from left (fig 2).
Step 3 - Heat fix - Heat fixation helps to fix samples to the glass slide and prevents sample loss during the staining process. Heat fixation of a slide can be done by a variety of methods. One method is to use a blow dryer directly on the slide from a distance of about 20-25cm. A Bunsen burner, a cigarette lighter or even a match can also be used. Hold the slide over the flame for 5 seconds without burning the slide or damaging the sample by overheating. If the flame is not burning “clean,” (as with a Bunsen burner) soot will accumulate on the bottom of the slide. This can easily be wiped off before the slide is stained.
Step 4 - The most commonly used stains in general practice for this purpose are the modified Wright’s stains, although some practices routinely perform a Gram stain. Note that bacteria always stain blue if using the Diff-Quik staining.
Step 5 – Microscopical examination – It is important to keep the light condenser up when examining a cytology sample. This is in contrast to examining skin scrapes or unstained tape strip samples where the shadows and contrasts created by keeping the condenser down can aid in the identification of fungal spores, hyphae, parasites, and hair shaft abnormalities.
The presence and number of microorganisms and inflammatory and/or epithelial cells helps in determining the presence of infectious agents and their role in the disease. The following organisms can be identified on cytological examination of dogs and cats with otitis externa: cocci (Staphylococcus, Streptococcus), rods (Pseudomonas, Proteus, and other Gram-negative bacteria), budding yeasts (Malassezia and Candida). The presence of white blood cells and phagocytosis of the bacteria in the otic discharge are signs that the body is responding to the infection (Fig 3).
Cytological preparations should first be scanned under low power for an area of evenly distributed cells, one layer thick. The use of a x100 oil immersion objective is then recommended for detailed examination of cellular morphology.
The diagnosis of a diseased external ear canal via cytology requires knowledge of the cytology of the normal external ear canal. A study was conducted on 50 normal dogs and 52 normal cats in order to characterize the normal cytological findings in specimens taken from the vertical ear canal by swabbing. Yeasts were detected in 96% of the dogs and 83% of the cats. Gram-positive cocci were found in 42% of the dogs and 71% of the cats. Rods were not seen. It has been reported that in normal dogs, the median numbers per high-power microscopic field (400· magnification) for yeast, cocci and keratinocytes should be 0.2, 0 and 3.9, respectively. In cats, the median numbers should be 0.2, 0.3 and 8, respectively.
Previous studies have determined that average otic bacterial counts greater than 25 organisms/HPF are significant. In another study cocci counts greater than 5/HPF or rod counts greater than 1/HPF should be considered pathogenic.
In the early stages of otitis externa resulting from atopic dermatitis, ear cytology may reveal the presence of large numbers of corneocytes but no evidence of infection, therefore antimicrobial therapy would not be indicated. Numerous yeast organisms will be seen in cases of Malassezia otitis externa. Cocci or rods will be evident in bacterial otitis and it is not uncommon to find several different microorganisms. Bacterial culture and sensitivity testing should be considered if cytology reveals the presence of rods. The presence of neutrophils and phagocytosed rods is commonly associated with Pseudomonas aeruginosa infection.
In a general practice situation, initial experiences, particularly with ear cytology, may be unrewarding. Nevertheless, with practice and increasing experience, the clinician should find these tests of great value in making a diagnosis. Discussing the prognosis, formulating a treatment plan and case management is immediately more rewarding. The ultimate result is significantly happier patients and clients.