If necessary, remove hair over areas to be sampled by careful clipping.

       Use a wooden tongue depressor for superficial sampling or a large, curved scalpel blade if deeper samples are required, with the sharp edge blunted to reduce the risk of injuring the horse or operator.

       Moisten the sample site or the collection tool with liquid paraffin (more useful for examination for mites), water or normal saline (for dermatophytes).

       Gently scrape crusts, scales, and associated hair so that the material accumulates on the blade or tongue depressor. Transfer onto a microscope slide with more liquid paraffin, or with potassium hydroxide solution if collected in aqueous medium, which allows clearing of debris and easier identification of pathogens.

       Deeper scrapings are needed for suspected demodicosis, deep enough to cause capillary ooze.

       Sample several sites, collect plenty of material, and divide amongst several slides to make thin suspensions, which are quicker and easier to examine efficiently.

       A piece of clear adhesive tape (e.g. 3M Scotch Crystal, Sellotape Clear) is applied to the lesional area 3–4 times (Figure 1.5).

       Tape is applied (sticky side down) to a microscope slide over a drop of liquid paraffin for direct examination or over a drop of blue dye from a rapid Romanowsky‐type stain kit.

       Excessive mounting medium or stain is removed by wiping with soft paper towel prior to microscopical examination.

       Press a glass slide against the concave undersurface of a removed exudative crust, or against the surface of a freshly exposed lesion.

       For an intact pustule, gently break the overlying skin with a 25 g needle and press a clean glass slide to the ruptured lesion, or purulent material may be collected in the needle bevel and then transferred to the glass slide.

       For lesions at sites where it is difficult to apply a slide directly, material can be collected with a dry swab and then rolled onto a glass slide.

       Air‐dry the slide and store in a slide box prior to heatfixing (for Gram staining) or immersion in methanol (for Romanowsky‐type staining).

       Representative sample of crust is placed on a glass slide with a few drops of normal saline.

       Material is finely chopped and macerated with a scalpel blade.

       Slide is left in a warm place for 20–30 min to allow rehydration of cellular material.

       Any large clumps of debris are gently removed prior to thorough drying and heat fixing of the remaining suspension prior to staining with a rapid Romanowsky‐type stain kit.

       If the sample is to be processed within 30 min of collection, a dry, sterile swab can be used for bacterial and fungal culture, and smears. Otherwise, place swabs into suitable transport media (e.g. Amies charcoal medium, or Copan ESwabs).

       Samples collected from the skin surface may not be representative of the causative agent, so collect pus from an intact pustule or the underside of a freshly removed scab, or submit biopsy material for culture. Useful cultures may sometimes be obtained from a dry crust by rehydrating with sterile normal saline prior to processing.

       A 20–22 g needle can be used, with or without a 5 ml syringe. The area to be aspirated should be carefully cleaned and disinfected.

       The needle is inserted into the nodule (Figure 1.6), mass, or lymph node and used to probe the tissue in several places, initially without aspirating, and subsequently whilst gently aspirating.

       The needle is withdrawn from the tissue and detached from the syringe, which is then filled with a small amount of air, reattached to the needle, and the sample expressed directly onto a clean slide for cytology or onto a swab for culture. A second slide is placed over the sample to spread the material. The slides are then separated gently to avoid damaging cells and air‐dried prior to staining for microscopical examination.