Page 269 - The Veterinary Laboratory and Field Manual 3rd Edition

238 Susan C. Cork and Roy Halliwell

The direct microscopic examination of hair a light source is used the contrast will not be
specimens, with or without culture, is the rec- adequate and the fungi will not be seen.
ommended method for detecting the presence of
dermatophytes.
Appearance of skin scales, nails or
crusts in KOH preparation
Direct microscopy
Look for branching hyphae, chains of angular
Fungi are usually larger than bacteria and can or rounded arthrospores (spore resulting from
readily be seen by direct microscopy provided hyphal fragmentation) or a mixture of both. All
the material from skin, hair or nails is first species of ringworm fungi have a similar appear-
softened and cleared with a strong alkali such ance. Ringworm fungi must be distinguished
as 200 g/1 (20% w/v) potassium hydroxide from epidermal cell outlines, elastic fibres and
(KOH). The purpose of the alkali is to digest the artefacts such as intracellular cholesterol and
keratin surrounding the fungi in samples so that strands of cotton or vegetable fibres. Fungal
the hyphae and spores can be seen. The method hyphae can be differentiated from these struc-
of examining a KOH preparation is as follows. tures by their branching growth, uniform width
Place a drop of 10–20% potassium hydroxide and cross-walls (septa) which can be seen when
(KOH) solution and sample on a slide. Proceed using the 40× objective.
with caution as the KOH solution is corrosive.
Mix with a drop of blue-black fountain pen ink Culture
or lactophenol blue and put a coverslip over the
preparation. The dye will highlight morpho- Identification of fungi is a specialist job. It should
logical features but these are not necessarily also be noted that 10–20% of specimens collected
diagnostic for specific fungi. Place the slide in a from ringworm lesions, where fungi are seen micro-
Petri dish or other container with a lid, together scopically, are negative on culture. This is usually
with a damp piece of filter paper or cotton wool due to the material collected being non-viable. In
to prevent the preparation from drying out. addition, contaminating bacteria or non-pathogenic
Note: To assist clearing, hairs should not be fungal spores in a specimen may overgrow patho-
more than 5 mm long and skin scales, crusts and genic species if steps are not taken to limit or
nail (horn/hoof) snips should be not be more remove them before attempting fungal culture.
than 2 mm in diameter. Isolation of ringworm and other fungal patho-
Hairs will clear within 5–10 min. Skin scales gens can be achieved by:
and crusts usually take 20–30 min. Pieces of
nail, however, may take several hours to clear. 1 adding inhibitory substances to the medium
Clearing can be hastened by gently heating the (see below)
preparation over the flame of a spirit lamp or 2 leaving the specimen to dry out, so contami-
pilot flame of a Bunsen burner taking care to pre- nating bacteria, but not fungi, will die off
vent drying or splashing of the corrosive KOH 3 adding acetone or alcohol to the specimen
solution (wear protective glasses when perform- and culturing after a few minutes.
ing this procedure).
As soon as the specimen has cleared, examine Choice of media
it microscopically using 10× and 40× objectives
with the condenser iris diaphragm closed suf- 1 Blood agar with added antibiotics to inhibit
ficiently to give a good contrast. If too intense bacterial growth.

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