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Cytology
SKIN SCRAPING
Skin scraping can be used for ectoparasites and dermatophytes. Supercial Skin Scrapes are suitable for scabies and short body Demodex mites. Deep Skin Scrapes are needed for long body Demodex mites. Start by clipping a large area of hair at the site of interest. Apply a thin layer of parafn oil. If required, sample 25 separate areas. For a Supercial Skin Scrape, use a scalpel blade to scrape all scale in a wedge to a collection point. Sampling should not cause bleeding. For a Deep Skin Scrape, lift the skin and squeeze rmly. Sampling should result in capillary bleeding. The parafn and expressed material should be collected. For both methods, the sample should be mixed with a drop of parafn oil on a glass slide. A cover slip should be applied and the slide examined under low power (x 40 or x 100 magnication).

Cytology is a quick and easy tool in the diagnosis and management of otitis and skin infections, allowing the presence of organisms to be determined by microscopic examination of prepared slides. This process ensures more accurate targeting of the causal organisms and, as a result, a faster resolution rate. Cytology only takes a few minutes and provides invaluable information that is helpful for both initial assessment and in monitoring the success of therapy.

TAPE SAMPLING
Adhesive tape sampling is ideal for sampling greasy or scaly lesions. It is commonly used to nd Malassezia and bacteria, but mites and lice may also be seen. Use a good quality, clear sticky tape and press rmly against the skin. Repeatedly press the tape onto the area to be sampled until the tape is no longer sticky. Fasten the sticky end of the tape to a glass slide, then add a drop of purple DiffQuik or RAPIDIFF stain and stick the tape down over it. Squeeze out the excess stain and blot away. Alternatively, stain using the conventional DiffQuik or RAPIDIFF method. Examine under oil immersion (x 1,000 magnication), using the tape as a cover slip. Image and text courtesy of Dermcare-Vet, Australia.

COTTON SWAB SAMPLING

It is easy to obtain a sample from the ear by gently removing the otic discharge with a cotton bud. Cotton bud sampling is also suitable for collecting samples from the skin and in areas such as between the toes. Warning! Cotton buds should not be inserted into ears for any reason other than for collecting samples. This could lead to damage to the lining of the ear canal and/or push ceruminous material down toward the tympanic membrane. Tip: If ear mite infestations are suspected, an additional slide should be prepared and examined microscopically, unstained and under low power.

PREPARING THE SMEAR

The material should then be gently transferred onto a microscope slide by rolling the cotton bud along the glass surface. Do not rub the swab backwards and forwards because this will disrupt the cellular components. Tip: If marked properly, material from both ears can be spread onto the opposing sides of a single slide, saving time and slides.

FIXING THE PREPARATION

STAINING THE SLIDE

MICROSCOPY
Examine under a microscope, starting with the lowest magnication. Initially, choose an area showing copious purple material, then use the lenses with higher magnication. Closely examine under the oil immersion lens (x 1,000 magnication) to search for cells and infectious organisms.

INTERPRETING THE SLIDES

Cells (x 400)

Yeasts

Cocci (x 2,000)

Rods (x 2,000)

Depending on the quality of the material, the slide should be air-dried (purulent material) or gently heat xed with low heat (waxy/oily discharge). Tip: A hairdryer can be used for gentle heat xation and will also be useful for drying the slide after rinsing. See STAINING THE SLIDE .

Stain the slide, using a modied Wrights staining system such as DiffQuik or RAPIDIFF, according to the manufacturers instructions. Dip 5-10 times in each solution, starting with xative solution, followed by stain solution 1 (red eosin dye) and stain solution 2 (blue thiazine dye or methylene blue dye). Rinse with water, then air-dry or dry gently with a hairdryer. Warning: Wear gloves or use forceps to protect your hands from long-lasting stains.

Tip: To avoid light scatter, which would cause slight blurring of the image, apply a drop of mineral or immersion oil over the stained material. Cover with a cover slip and then examine microscopically.

Interpretation of cytological slides requires some experience and practice. Examine the sample under high-power x 400 magnication to detect inammatory cells (mainly neutrophils and macrophages) and under x 2,000 magnication with oil immersion to detect cocci, rods and yeasts. Tip: Make sure you dont confuse cocci (perfectly round, similarly sized and purple in colour) with melanin granules (black/brown/yellow colour) or irregularly shaped stain aggregates, when focusing up and down.

Typical Results
NORMAL CYTOLOGY
It is easier to decide what is abnormal after having examined cytology slides from a few normal sites. Occasional cocci and yeasts are within normal limits, but most elds will only contain translucent, large, polygonal cells without a nucleus i.e. keratinocytes or skin cells. Some of these keratinocytes are stained purple and may be cigar shaped. Tip: You should send duplicates of your slides to an experienced cytologist when starting out, and compare the cytology report with your own ndings.

IMPORTANT: Although the colour, consistency and smell of the otic discharge can give you some idea of what to expect, mere visual and olfactory examination cannot replace cytology.

BACTERIAL INFECTION/OVERGROWTH
The photomicrograph shows degenerate neutrophils with intra- and extracellular cocci diagnostic for bacterial infection, most likely associated with Staphylococcus pseudintermedius. Bacterial overgrowth with numerous cocci in the absence of any inammatory reaction can sometimes be found. Tip: Although antibiotic therapy improves the condition, it is important to identify and treat the underlying disease.
Neutrophil with engulfed cocci

MALASSEZIA SPP.
Malassezia pachydermatis yeast is a very common causal organism, often isolated alongside Staphylococcus pseudintermedius (x 2,000 magnification). Note the peanut/footprint/ snowman/Russian doll shape of the yeast. Malassezia pachydermatis is commonly found in patients with atopic dermatitis, but also in other otitis cases. The discharge is often brown/waxy and has a characteristic yeasty odour.
Bacterial overgrowth

PSEUDOMONAS SPP.
Whenever rod shaped bacteria are seen on cytology, bacterial culture should be performed, because the organisms could be Pseudomonas aeruginosa. Infections with Pseudomonas spp. may require intense medical therapy and carry a cautious prognosis. The antibiotic chosen should always be based on sensitivity testing. It is prudent to reserve some types of antibiotics for conditions which have responded poorly, or are expected to respond poorly, to other classes of antibiotics.

Keratinocyte (cigar shaped)

Although some yeast organisms can be found in healthy ears, treatment is advised even when relatively small numbers of yeast cells can be seen, if the clinical signs (erythema and/or pruritus) suggest they play a role in the disease.

Dechra Veterinary Products Ltd would like to thank Ariane Neuber (DrMedVet, CertVD, DipECVD, MRCVS) and Dermcare-Vet, Australia for their help in preparing this information sheet.

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CANAURAL: Each g of Canaural contains Fusidic acid (diethanolamine fusidate) 5 mg, framycetin 5 mg, nystatin 100,000 units, prednisolone 2.5 mg. UK: POM-V IE: POM FUCIDERM: Fuciderm Gel contains 0.5% w/w Fusidic acid and 0.1% w/w betamethasone valerate. UK: POM-V IE: POM MALASEB: Malaseb Shampoo contains 2% w/v chlorhexidine and 2% w/v miconazole. UK: POM-V IE: POM Further information is available on request from: Dechra Veterinary Products Ltd, Cartmel Drive, Harlescott, Shrewsbury, Shropshire SY1 3TB. Tel: +44 (0)1743 441632. Fax: +44 (0)1743 462111. Dechra Veterinary Products Ltd is a trading division of Dechra Ltd. Registered Ofce: Dechra House, Jamage Industrial Estate, Talke Pits, Stoke-on-Trent, Staffordshire ST7 1XW Registered in England No: 5385888. www.dechra.com/derma .