References

Duncan D. Use of cytology for diagnostics in veterinary dermatology. The Veterinary Nurse. 2011; 2:(7)368-76

Forsythe P. Acute otitis externa: the successful first-opinion ear consultation. In Practice. 2016; 38:(2)2-6

Hill PB, Lo A, Eden CA Survey of the prevalence, diagnosis and treatment of dermatological conditions in small animals in general practice. Vet Rec. 2006; 158:533-9

Miller W, Griffin C, Campbell K. Diseases of eyelids, claws, anal sacs, and ears, 7th Ed. Missouri: Elsevier; 2013

Shaw S. Pathogens in otitis externa: diagnostic techniques to identify secondary causes of ear disease’. In Practice. 2016; 12-16

Toma S, Cornegliani L, Persico P, Noli C. Comparison of 4 fixation and staining methods for the cytologic evaluation of ear canals with clinical evidence of ceruminous otitis externa. Vet Clin Pathol. 2006; 35:(2)194-8

How to perform ear sampling and undertake microscopy

02 June 2017
Practical
5 mins read
02 June 2017
Volume 8 · Issue 5

Abstract

Otitis externa is a common presentation in small animal veterinary practice and the veterinary nurse can play a vital role in managing these cases. Ear sampling techniques are relatively straightforward and together with microscopic examination form two vital investigative processes in which veterinary nurses can become competent.

Otitis externa is a common presentation in small animal veterinary practice (Hill et al, 2006). The veterinary nurse plays a vital role in helping the veterinary surgeon to examine the ear, take samples and perform microscopy. This not only helps the smooth running of a busy veterinary practice, but also ensures prompt and correct treatment for the patient, keeping both patient and owner content. Ear sampling is fairly simple, quick and inexpensive, the equipment needed is readily available, and in-house assessment usually works out cheaper than sending the sample to an external laboratory. Ear sampling should be performed for every patient presenting to the practice with otitis, but it is also essential at each follow-up appointment to ensure treatment is effective. To facilitate sampling and assessment the veterinary nurse can ask the owner to cease any ear treatments 24–48 hours prior to the appointment.

Otitis externa can result from numerous underlying causes, e.g allergy, foreign bodies and ectoparasites to name a few (Miller et al, 2013) and ideally otoscopy should be performed prior to sampling (if the patient permits). This is for several reasons:

  • To check for a foreign body before inserting the cotton bud
  • To check the integrity of the tympanic membrane; in case inserting the cotton bud dislodges any discharge and obscures the view
  • To check for erythema/ulcers/erosions; in the author's opinion, if the ear canal is friable inserting the cotton bud to collect a sample can cause trauma and in some cases, haemorrhage.

    • Equipment needed

  • Microscope slides with frosted ends for easy labelling
  • Coverslips
  • Liquid paraffin
  • Cotton buds
  • Pencil
  • Immersion oil
  • Modified Wright's stain such as DiffQuik™ (or similar) (Figure 1)
  • Microscope.
    • Figure 1. Ear sampling equipment.

    Step-by-step guide

  • Prepare all equipment (Figure 1) — this is very important especially if the patient is fractious.
  • Restraint — restraint can be the difference between getting the sample and not. Adequate restraint of the patient to keep the head still will minimise any pain the patient may experience during sampling and permit full assessment of the ear canal.
  • Perform indirect impression smear (and/or culture and susceptibility testing) — the cotton bud is used to collect the sample from the vertical canal (Figure 2). In some instances it can be difficult to sample the ear with a cotton bud, due to the patient being head shy. In these circumstances, it is sometimes easier to gently insert a gloved finger into the ear canal to collect the sample (Forsythe, 2016) (Figure 3). Culture and susceptibility testing is performed depending on the results of cytology and determines whether systemic antibiotics are required.
  • Both ears should be sampled as the findings from each ear may differ.
    • Figure 2. Collecting the sample.
    Figure 3. Using a gloved finger to collect the sample.

    Be aware that not all patients will tolerate examination and sampling of the ear while conscious; whether this is due to pain or being head shy due to chronic ear problems. These patients may need to be sedated and/or anaesthetised to allow a thorough examination to be performed and should be discussed with and instructed by the veterinary surgeon.

    Preparing an indirect impression smear for cytology

  • Label the slide with the patient's name and ear (left or right) with the pencil (Figure 4).
  • Gently roll the cotton bud over the slide (Figure 5). If sample was collected with a gloved finger, press the finger directly onto the slide, but do not smear the sample.
  • Allow the sample to dry for a few minutes or until it looks dry.
  • Once the sample is dry, stain the slide using a modified Wright's stain (Figure 6). If the sample looks purulent, dip the slide 5–10 times in each stain, rinsing gently at the end with water. If the sample looks waxy, miss out the fixative (blue) as this may dissolve the sample and proceed to 5–10 dips in the eosinophilic (red) stain followed by 5–10 dips in the basophilic (purple) stain before rinsing gently with water. It has been suggested that these samples should be heat fixed, but this has been shown to be unnecessary (Toma et al, 2006).
  • If there is time, allow the stained slide to dry, however if time is limited gently blot any excess liquid away taking care not to rub the slide as this may disturb the smear created.
  • Once dry, apply a drop of either liquid paraffin or immersion oil onto the sample and cover with a cover slip (microscopes are designed to look through a fluid layer, this will give you a clearer image and keep your microscope clean).
  • The slide is now ready for examination under the microscope.
    • Figure 4. Label slide.
    Figure 5. Roll the sample onto a dry microscope slide.
    Figure 6. Modified Wrights Stain, e.g DiffQuik™ station.

    Preparing a slide for assessment for ectoparasites

  • Label slide with the patient's name and ear (left or right) with the pencil (Figure 4).
  • Apply a drop of liquid paraffin (Figure 7).
  • Roll and emulsify the cotton bud in the liquid paraffin to transfer the sample, ensuring the sample is spread evenly through the liquid paraffin (Figure 8).
  • Apply a cover slip (Figure 9) — cover slips are essential to ensure the sample is evenly dispersed.
  • The slide is now ready for examination under the microscope.
    • Figure 7. Liquid paraffin prepared for sample.
    Figure 8. Roll the cotton bud in liquid paraffin.
    Figure 9. Apply a coverslip.

    Examining the sample

    When examining slides the Vernier scale should be used to map out findings to ensure the veterinary surgeon can re-locate and verify the findings if needed. It is important to remember when using the Vernier scale the same microscope must be used, the slide placed in the original position on the stage, and both the horizontal and vertical scales are recorded.

    Assessment for ectoparasites

    The slide should be examined under low power with the iris diaphragm closed (Figure 10). The sample should be examined for Otodectes or Demodex mites. Closing the iris diaphragm increases contrast and helps to identify the mites. The slide should be examined using the ‘castle-top’ method to ensure the whole slide is viewed systematically. All ectoparasites are visible at x4 magnification and this will enable the slide to be viewed more quickly; however x10 may be needed to determine if the mite is alive.

    Figure 10. Iris diaphragm.

    Cytology

    This slide should be examined under low power initially (x4 or x10) with the iris diaphragm open (Figure 10). Little detail will be visible at low magnification but it is possible to determine whether the sample is homogenous (uniform) or heterogeneous (non-uniform) which identifies the areas of interest for closer inspection. Once an area of interest is found the magnification can be increased (Shaw, 2016) (x40 and then x100 oil immersion) to look for yeasts, bacteria and inflammatory cells.

    Should culture and susceptibility testing be performed?

    Culture and susceptibility testing is generally not required for the investigation of otitis externa as most cases are treated with topical drops and susceptibility results apply to anti-microbials that are used systemically. It may, however, be indicated in certain situations in order to confirm the species of bacteria, particularly with increasing antimicrobial resistant bacterial carriage and infections in patients. For assessing response to treatment, clinical improvement and repeat cytology should be used to guide further treatment rather than repeat culture as results can be misleading once topical therapy has been used.

    Conclusion

    Time pressures of a busy veterinary clinic are the most common reason why ear sampling and in-house examination are not performed. Ear sampling techniques are relatively straight-forward and together with microscopic examination form two vital investigative processes in which veterinary nurses can become competent with regular practice. The equipment needed is readily available and inexpensive (Duncan, 2011). Rapid diagnosis and implementation of treatment together with owner compliance will therefore result in a more positive and cost-effective outcome.

    Key points

  • Otitis externa is a common presentation in small animal veterinary practice and the veterinary nurse can play a vital role in helping with these cases.
  • For efficiency a sampling box/tray should be prepared and regularly re-stocked with all equipment needed to perform ear sampling.
  • Practice makes perfect — to become competent with the skills involved in ear sampling and microscopy they should be practiced as often as possible.
  • Using the in-house laboratory can result in a rapid diagnosis and usually is cheaper than sending to an external laboratory.
    • otitis
    microscopy
    veterinary nurses