Research in VeterinaryScience 1996, 61, 273-274 Comparison of media and conditions of incubation for the quantitative culture of Malassezia pachydermatis from canine skin R. BOND, D. H. LLOYD, Dermatology Unit, Department of Small Animal Medicine and Surgery, The Royal Veterinary College, University of London, Hawkshead lane, North Mymms, Hatfield, Herts AL9 7TA SUMMARY Fifty-five swab-wash specimens from dogs were cultured at 32°C on Sabouraud's dextrose agar either with or without 1 per cent Tween 80, Ushijima's medium A and modified Dixon' s and Leeming's media. The counts of Malassezia pachydermatis were not significantly different after three or seven days of incubation. Colony counts on contact plates were significantly greater after incubation for seven days on Sabouraud's dextrose (P<0.001) and modified Dixon's agars (P<0.05) than after three days at 26°C, but not at 32°C. Counts on Sabouraud's or modified Dixon's agars after incubation at 32°C and 37°C were not significantly different. When compared with aerobic culture, an atmosphere containing 5 to 10 per cent carbon dioxide significantly (P<0.01) increased the frequency of isolation and colony counts on Sabouraud's dextrose agar but not on modified Dixon's agar. QUANTITATIVE techniques suitable for counting Malassezia pachydermatis on canine skin are required to study the biology of this yeast in health and disease. In quantitative investigations of the human skin flora the culture medium has been shown to have significant efforts on the counts of bacteria (Noble and Somerville 1974) and the lipid-dependent yeast Mfurfur (Faergemann 1987, Leeming and Notman 1987, Korting et al 1991). A milk-contain- ing medium (Leeming and Notman 1987) yields higher counts of M furfur colonies than modified Dixon's agar (Midgley 1989, van Abbe 1964) or an olive oil-containing medium (Faergemann and Fredriksson 1980, Korting et al 1991). M pachydermatis is less fastidious and grows on various media without lipid supplementa- tion (Fraser 1961, Yarrow and Aheam 1984, Ushijima et al 1981), both aerobically and in an atmosphere containing 7 to 10 per cent carbon dioxide (Fraser 1961, Faergemann and Bernander 1981). Leeming's medium and modified Dixon's agar also support the growth of Mpachydermatis but the value of these and other media for the isolation of this yeast does not appear to have been deter- mined; this was the purpose of the work reported in this study. Five media were used: Sabouraud's dextrose agar (Oxoid CM41, 65 g litre-t); Sabouraud's dextrose agar containing 1 per cent Tween 80 (BDH); modified Dixon's agar (Bond et al 1994); Ushijima's medium A (Ushijima et al 1981) and Leeming's medi- um (Leeming and Notman 1987). Tryptone (Oxoid L42) was sub- stituted for trypticase peptone (BBL) in Ushijima's medium and agar No 3 was substituted for agar No 1 in Ushijima's and Leeming's media. The media were poured into sterile 90 mm petri dishes, kept at 4°C and used within seven days. The dogs were sampled using either contact plates (Bond et al 1994) or a semi-quantitative swab technique (Allaker et al 1992, Bond et al 1995b). Contact plates were made by filling 28 mm universal bottle lids with modified Dixon's agar or Sabouraud's dextrose agar, and the skin was sampled by applying these plates for 10 secoJads. Other sites were swabbed for five seconds with sterile cotton-wool swabs, the tips of which were cut off into ster- ile bijou bottles containing 2 ml of a 'wash fluid' composed of sterile 0.075M phosphate-buffered saline (pH 7.9) containing 0.1 per cent Triton X-100 (13DH). The bottles were then shaken manu- ally for 30 seconds. Counts of viable M pachydermatis were obtained by spreading 25 pl aliquots of the wash fluid samples, and serial 10-fold dilutions of the fluid, where appropriate, on to paired quadrants of duplicate agar plates within 40 minutes of col- lection (Bond et al 1995a). The inoculated plates were incubated as described below. M pachydermatis was identified by its gross colonial and midroscopic morphology. In addition, Malassezia species isolated on Sabouraud's dextrose agar containing 1 per cent Tween 80, modified Dixon's agar and Leeming's medium were identified as M pachydermatis if they were able to grow when transferred on to Sabouraud's dextrose agar (Gordon 1979, Midgley 1989). Counts derived from the swab and contact plate techniques were expressed as log10 colony forming units (cfu) per swab and log10 cfu per plate. A total of 55 swabs was obtained from the left and right exter- nal ear canal and the anus of 15 healthy beagles and from either the left or right external ear canal of 10 dogs with erythematoceru- ruinous otitis externa. Each wash-fluid sample was inoculated on to the five media as previously described. Plates were incubated aerobically at 32°C and cfu were determined after three and seven days of incubation. M pachydermatis was isolated on each of the five media. After three days of incubation, the colonies were most distinctive on modified Dixon's agar, forming buff-coloured, domed colonies 1 to 1.3 mm in diameter which were usually readily distinguishable from the other cutaneous microbes; on the other media the colonies were convex. The counts obtained on the five media did not differ significantly after either three or seven days of incuba- tion at 32°C (Table 1). For each medium, the paired counts at three and seven days were also not significantly different. The contact plates were used to sample four skin sites on each of seven dogs with dermatitis associated with M pachydermatis. Two plates containing Sabouraud's dextrose agar and two con- taining modified Dixon's agar were applied to each site in random order and incubated at 26°C and 32°C. Counts of M pachyderma- tis colonies were made three and seven days later. Preliminary studies had indicated that the counts obtained from four succes- sive applications of contact plates at 32 sites on 11 dogs with der- matitis associated with the yeast did not vary significantly. When incubated at 26°C, the mean (sE) counts (log10 cfu per plate) after seven days on Sabourand's dextrose agar (1.2 [0.2]) and modified Dixon's agar (1.8 [0.2]) were significantly greater than the three-day counts (0-8 [0.1], P<0.001 and 1.5 [0.2], P<0-05, respectively [paired t test]). When incubated at 32°C, the TABLE 1: Mean (SE) Malassezia pachydermatis counts (log10 cfu per swab) on five different media after three and seven days of incubation at 32°C Medium Day 3 Day 7 Sabouraud's dextrose agar ° 1.9 (0-2) 1.9 (0-2) Sabouraud's dextrose agar plus 1% Tween 80 2.0 (0.2) 1.8 (0.2) Ushijima's medium A 1.8 (0.2) 1-8 (0.2) Modified Dixon's agar 2.0 (0-2) 2-0 (0.2) Leeming's medium 2.1 (0.2) 2-1 (0.2) 273