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1 Department of Microbiology, Faculty of Medicine, University of Çukurova, Adana, Turkey
2 Department of Microbiology, Faculty of Medicine, University of Ege, Izmir, Turkey
Correspondence
Macit Ilkit
milkit{at}cu.edu.tr
Received 12 March 2006
Accepted 8 February 2007
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONREFERENCES |
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Chromogenic media such as Albicans ID2 and Biggy agar allow rapid and presumptive identification of yeasts on primary plates. An indicator substance in these media allows the differentiation of different species according to their colony colour or morphology (Lipperheide et al., 1993; Silva et al., 2004). Bismuth sulfite glucose glycine yeast or Biggy agar (Oxoid), also known as Nickerson’s medium, is a selective and differential medium used in the detection, isolation and presumptive identification of Candida species. The bismuth sulfite acts as an inhibitory agent to suppress bacterial growth, which enables the recovery of isolated colonies of Candida. Candida species produce sulfide and bismuth, which combine to produce brown to black-pigmented colonies and zones of dark precipitate in the medium surrounding colonies of some species through a process of substrate reduction. Candida albicans, Candida tropicalis, Candida krusei and Candida kefyr were reported to be identifiable on Biggy agar by the manufacturer (Nickerson, 1953; Mendel et al., 1960; Kalkanci et al., 1999; Aktas et al., 2001; Yucesoy & Marol, 2003; Silva et al., 2004; Yucesoy et al., 2005).
Albicans ID2 (bioMérieux) contains a chromogenic substrate for the enzyme hexosaminidase with chloramphenicol and gentamicin. Hydrolysis of this substrate, which is characteristic of C. albicans, leads to the appearance of blue-pigmented colonies, whilst non-albicans Candida strains remain white (Lipperheide et al., 1993; Rousselle et al., 1994; De Champs et al., 1995; Baumgartner et al., 1996; Contreras et al., 1996; Freydiére et al., 1997; Hoppe & Frey, 1999; Ilkit et al., 1999; Fricker-Hidalgo et al., 2001; Godoy et al., 2001; Cárdenes et al., 2002, 2004; Yucesoy et al., 2005). Albicans ID2 has been improved and the new plates are known as Candida ID2. They have two chromogenic substrates and give blue colonies for C. albicans, pink colonies for certain non-albicans Candida species such as C. tropicalis, Candida lusitaniae and C. kefyr, and white for the rest.
The aim of this study was to compare the performance of the chromogenic media Albicans ID2 and Biggy agar with that of a reference medium, Sabouraud chloramphenicol agar (SCA), for rapid and non-microscopic presumptive identification of yeast species obtained directly from clinical specimens, as well as to discuss the efficacy of chromogenic media in the mycology laboratory by using our data from 1998.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Procedure. Only one sample was examined from each patient. Vaginal samples were examined in order to determine the effectiveness of Albicans ID2 and Biggy agar for the growth and identification of Candida species in comparison with the routinely used SCA. Samples were taken with sterile swabs using a dry sterile speculum and inoculated into a Sabouraud 2 % glucose broth with 1 ml liquid glucose for their transport to the laboratory and kept in this broth for a maximum of 30 min before inoculation of plates by streaking the swab.
Commercial media. Biggy agar and Sabouraud agar (Difco Laboratories) were purchased as powdered media and prepared according to the manufacturer’s instructions in Petri dishes containing 15 ml of the liquid medium. Chloramphenicol (100 µg ml–1) was added to the Sabouraud agar and the media were stored at 4 °C until use. Albicans ID2 is a commercial ready-to-use medium.
Evaluation of the media. All specimens were inoculated onto Albicans ID2, Biggy agar and SCA in parallel. After inoculation, the Albicans ID2 and SCA cultures were incubated at 37 °C, whilst Biggy plates were incubated at 30 °C, according to the manufacturers’ instructions. Interpretation of the results was carried out after 24 and 48 h of incubation.
Identification.
All yeast isolates observed on Albicans ID2 and Biggy agar were identified by colony pigmentation and morphology according to the manufacturers’ instructions. The criteria for the identification of C. albicans on the two chromogenic media were any blue colour (including weak blue) on Albicans ID2 and a dark brown–black colour with a yellow zone on Biggy agar. All colonies on SCA were identified by conventional methods, i.e. germ-tube production in human serum at 37 °C for 2 h, micromorphology on cornmeal Tween 80 agar according to the Dalmau method and the commercial API 20C AUX kit (bioMérieux) (Buckley, 1989). Candida dubliniensis was differentiated from C. albicans by abundant chlamydospore formation on cornmeal Tween 80 agar, the absence of fermentation of xylose and methyl 
-D-glucoside on API 20C AUX and the absence of growth at 45 °C. The reference strains C. albicans ATCC 10231 and C. tropicalis ATCC 22019 were used as quality controls.
Statistical analysis. The sensitivity and specificity of the colony appearances of C. albicans on Albicans ID2 and Biggy agar were determined as the number of true positives/(number of true positives+number of false negatives) and the number of true negatives/(number of true negatives+number of false positives), respectively.
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The results were the same after 24 and 48 h of incubation for both media. Mixed fungal cultures and bacterial growth or filamentous fungi were not detected on either of the chromogenic media or SCA.
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On Biggy agar, C. kefyr colonies were easily distinguished from those of the other species by their reddish-brown colour, medium size, straight surfaces and projected edges. C. albicans and C. dubliniensis colonies were distinguished from the other species by the formation of dark brown–black colonies and small mycelial fringes, but it was not possible to differentiate them from each other. C. tropicalis, C. krusei and C. parapsilosis formed dark-brown colonies with slight mycelial fringes, but it was difficult to distinguish them from each other, as well as from the other species. However, whilst 10 of the 20 C. glabrata colonies formed brown colonies with yellow zones, 10 formed light-brown colonies with yellow zones, in addition to the two C. guilliermondii strains with brown colonies surrounded by yellow zones, and hence they could not be distinguished from each other. The sensitivity and specificity of Albicans ID2 and Biggy agar with regard to the identification of C. albicans were 80.0 and 64.6 %, and 86.7 % and 56.3 %, respectively.
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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For the rapid identification of C. albicans, the sensitivity and specificity of Albicans ID2 have been estimated as 92.7–100 and 86.6–100 %, respectively (Lipperheide et al., 1993; Rousselle et al., 1994; De Champs et al., 1995; Baumgartner et al., 1996; Contreras et al., 1996; Freydiére et al., 1997; Hoppe & Frey, 1999; Ilkit et al., 1999; Fricker-Hidalgo et al., 2001; Godoy et al., 2001; Cárdenes et al., 2002, 2004; Yucesoy et al., 2005). In our study, the sensitivity of Albicans ID2 medium was 80.0 % and the specificity was 64.6 %, much lower than the results of the studies mentioned above. This may be due to the lower isolation rate in the present study. On the other hand, a surprising result obtained in our study was the isolation of a panel of Candida species with C. glabrata as the primary species. More recently, Niemann et al. (2005) reported the detection of Candida species in 173 out of 600 vaginal samples (28.8 %), with 85.5 % of them being C. albicans, 8.7 % C. glabrata, 1.2 % C. krusei, 0.6 % C. dubliniensis and 4 % other Candida species. The authors had isolated C. dubliniensis only once, from a non-symptomatic, HIV-seronegative pregnant proband.
Recently, it was reported that Albicans ID2 medium did not enable differentiation between C. albicans and C. dubliniensis, correlating with our study (Fricker-Hidalgo et al., 2001; Godoy et al., 2001). In contrast, Cárdenes et al. (2002) noted that they managed to distinguish all 38 C. dubliniensis strains on Albicans ID2 medium. In other studies, C. tropicalis was the main source of false-positive identification on this medium (Lipperheide et al., 1993; Rousselle et al., 1994; De Champs et al., 1995; Baumgartner et al., 1996; Hoppe & Frey, 1999; Ilkit et al., 1999; Fricker-Hidalgo et al., 2001; Godoy et al., 2001; Cárdenes et al., 2002, 2004). In accordance with these findings, in 10 C. tropicalis strains we observed two producing blue colonies and five producing weak-blue colonies as a false-positive result, whilst only three strains produced white colonies as a true-positive result. In addition, five C. glabrata and one C. krusei produced blue or weak-blue colonies and could not be distinguished from C. albicans (Table 1
).
Mendel et al. (1960) reported that bacterial inhibition by Biggy agar was superior to that of Pagano–Levin agar in the identification of vaginal samples. Kalkanci et al. (1999) determined that C. kefyr and Candida rugosa strains were easily distinguished from other species, but the colonies produced by such species had no features that could be used for identification on Biggy agar. In a study by Aktas et al. (2001), it was stated that C. albicans, C. kefyr and C. glabrata colonies were easily identified because of their different characteristics.
Yucesoy & Marol (2003) reported that it was difficult to differentiate the light or light and/or dark-brown colours of C. albicans and C. tropicalis, respectively. In their study, only two species showed a typical, distinctive appearance on Biggy agar. One of these was C. krusei, which produced typical large, rough, dark-brown colonies with a surrounding yellow zone, whilst the other was C. parapsilosis, which formed light brown–greenish, grey, cream-coloured colonies. In another study reported by Yucesoy et al. (2005), the sensitivity and specificity of CHROMagar Candida, Biggy agar and Albicans ID2 agar for the detection of C. albicans were 100 and 100 %, 91 and 92.7 %, and 99.2 and 92.7 %, respectively. The authors also reported that few isolates of C. parapsilosis and C. kefyr and all isolates of C. glabrata grew weakly on Biggy agar. The lower sensitivity and specificity of Biggy agar for the identification of commonly isolated Candida species potentially limits the clinical usefulness of this agar (Kalkanci et al., 1999; Aktas et al., 2001; Yucesoy & Marol, 2003; Yucesoy et al., 2005). Our results were found to be consistent with only two of the above-mentioned reports in terms of C. kefyr differentiation from other species; however, we could not identify features of other species (Kalkanci et al., 1999; Aktas et al., 2001).
This study reconfirms that both Albicans ID2 and Biggy agar are effective media for the isolation of yeast fungi. However, these two media could fail to identify the correct Candida species, e.g. three isolates of C. albicans produced white colonies on Albicans ID2 agar. In addition, in some cases, several Candida species could not be distinguished from each other on Biggy agar. In conclusion, the chromogenic media are not sufficient for reliable differentiation of yeasts to the species level and additional classical differentiation methods, e.g. subcultivation on rice agar, fermentation and assimilation techniques (e.g. ID 32C), are necessary to identify clinical yeast isolates.
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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erlendirilmesi. Turkiye Parazitol Derg 25, 301–304.
itli Candida kökenlerinin tiplendirilmesinde Biggy agar kullanilmasi. Infek Derg 13, 407–411.
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