JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1975, p. 354-358Copyright ©) 1975 American Society for Microbiology

Vol. 2, No. 4Printed in U.SA.

Evaluation of the Uni-Yeast-Tek Kit for the Identification ofMedically Important Yeasts

P. I. BOWMAN AND D. G. AHEARN*Department of Biology, Georgia State University, Atlanta, Georgia 30303

Received for publication 6 June 1975

The Uni-Yeast-Tek system, a commercially prepared kit and scheme for therapid identification of medically important yeasts (Corning Medical), was evalu-ated in comparison with a conventional procedure in the identification of 623yeasts. The system permitted the presumptive identification of 99.8% of 436isolates representing 16 common species commonly isolated in the clinicallaboratory. Correct biochemical and morphological analyses were obtained with48 other species, but their specific identification required additional data.

Detailed procedures for the identification ofyeasts have been described by Wickerham (15)and Lodder (8). Unfortunately, since thesemethods require a broad spectrum of tests,skilled interpretations, and time, they are em-ployed in few clinical laboratories. Adequateschemes for the selective identification of com-mon clinical isolates are available (1, 2, 11), butdue to lack of technical help and time eventhese simplified schemes rarely are used. Inhospitals, yeast diagnostic efforts consistmostly of tests for the detection of pseudomyce-lia and chlamydospores in Candida albicansand tests for capsule formation and urease activ-ity in Cryptococcus neoformans. These circum-stances together with the increased incidenceand importance of yeast infections have moti-vated several colnpanies to prepare yeast diag-nostic kits. This paper evaluates the Uni-Yeast-Tek system developed by Corning Medi-cal for the routine identification of clinicallyimportant yeasts.

MATERIALS AND METHODSSix hundred and twenty-three isolates, mainly

from clinical specimens, were selected from culturesreceived at the Mycology Division of the Center forDisease Control (CDC), Atlanta, and from the cul-ture collection at Georgia State University. All iso-lates were identified by conventional procedures (3)conducted mostly at CDC, and the data were madeavailable for comparison with results of the Uni-Yeast-Tek system. The conventional procedures in-cluded testing for formation of germ tubes on bovineserum and tissue culture medium 199 (BBL).The Uni-Yeast-Tek kit, received from Corning

Medical, Roslyn, N.Y., is composed of a multisec-tioned dish containing seven carbon assimilationagars (pH adjusted to 7.0), urea agar, nitrate assimi-lation agar (pH adjusted to 5.0), a central well con-taining corn meal with Tween 80 for mycelium andchlamydospore production, a broth containing 0.05%

glucose and 2.6% beef extract (Wilson Laboratories)for the production of germ tubes, instructions, and awheeled classification key for the identification of 16common clinical yeast isolates.

Commercially prepared Sabouraud dextrose agar(Difco), as recommended, was used to obtain colonyisolates of yeasts. Cells from a single colony weresuspended in 5 ml of sterile distilled water to give acell suspension of 106 to 107/ml (a 1+ reading on aWickerham card); 1 drop was used to inoculate eachassimilation agar via a Pasteur pipette through asmall portal on the side of each well. A secondSabouraud plate was used to determine growth at 37C.

To test for germ tubes, the glucose-beef extractbroth was inoculated by touching the tip of a pipettelightly to the surface of a colony and inserting thepipette into the broth and incubating at 37 C for 3 h.Germ tubes and all morphology were determinedmicroscopically. The central corn meal well was inoc-ulated with a standard Dalmau cut, and the platewas incubated at 22 to 26 C for 2 to 10 days. TheDalmau cut was examined microscopically on days 3and 5 after inoculation for observation of blasto-spore, pseudomycelium, chlamydospore, or arthro-spore production.The reaction media for the Uni-Yeast-Tek system

are listed in Table 1. The carbohydrates vary inconcentration from 1 to 4%, and the basal media areprepared in accordance with the formulas of Wicker-ham (15). Positive reactions on the assimilationagars are denoted by growth accompanied by colorchanges in the indicators, bromocresol purple for thecarbon assimilations and bromothymol blue in thenitrate medium. Agar reactions, i.e., color changesand growth, were read from 2 to 5 days.

RESULTSIn preliminary studies of varied inocula, in-

cluding cells starved on yeast nitrogen base andyeast carbon base for 48 h, cells starved for 1week in distilled water, and cells grown onSabouraud dextrose broth and washed twice insterile saline and resuspended to 108 cells/ml,

154

on May 30, 2018 by guest

http://jcm.asm

.org/D

ownloaded from

EVALUATION OF YEAST IDENTIFICATION SYSTEM

similar reactions were produced by representa-tive clinical isolates. Thereafter, all subsequenttesting employed the recommended inoculumpreparation.

All isolates of C. albicans and C. stellatoideawere readily distinguished from other yeasts bytheir germ tubes and assimilation spectra. Theglucose-beef extract broth provided with the kitproved comparable to tissue culture media 199and bovine serum in the production of germtubes. All isolates of C. albicans except threeproduced germ tubes within 3 h on the glucose-beef extract broth. Two cultures of C. albicansfrom patients who had received polyene ther-apy gave only sparse numbers of germ tubes inall preparations. The third isolate, Syringo-spora albicans (14), was somewhat atypical inall its morphological properties. A typical germtube produced by C. albicans is shown in Fig.la and b. Unlike pseudohyphal cells, germtubes are not constricted at their point of origin(3, 9). Pseudohyphal elements, which appeargrossly similar to germ tubes, may be producedby some isolates of C. tropicalis in the germtube test. Unlike germ tubes, these elongatedbuds are constricted at their point of origin(Fig. lc and d). It should be noted that some C.albicans may produce both germ tubes andpseudohyphal cells, but only the germ tubes arediagnostic.Of the 623 isolates examined, 436 were com-

mon clinical species whose identification pat-tern was provided in the Uni-Yeast-Tek classifi-cation scheme. The accuracy of identification of16 common clinical isolates as compared to con-ventional procedures is given in Table 2. Ofthese isolates, 99.8% were correctly identified.The one isolate of C. tropicalis that would notgrow on the Uni-Yeast-Tek media was CBS6418. In initial kits, isolates of this species weredistinguished on the basis ofcellobiose assimila-tion. Cellobiose assimilation by C. tropicalis,however, is frequently latent to negative onauxanographic procedures. In our preliminaryscreening of 47 isolates, all of which assimilatedcellobiose with prolonged incubation in liquidmedium, only 17 gave positive reactions withthe kit by 8 days. Thereafter the company modi-fied the kit to include soluble starch (Difco). Allisolates of C. tropicalis, including the typestrain deposited at the Centraalbureau voorSchimmelcultures (CBS), Delft, Holland (D.Yarrow, personal communication), and over100 cultures in our collection assimilated solu-ble starch, whereas isolates of C. parapsilosisin these same collections failed to utilize starch.The positive assimilation reactions for isolates:f C. tropicalis on soluble starch were rapid,

TABLE 1. Media and reactions of the Uni-Yeast-Teksystem

Reaction colorReaction media

Positive Negative

Urea Pink StrawSucrose Yellow PurpleLactose Yellow PurpleMaltose Yellow PurpleRaffinose Yellow PurpleCellobiose Yellow PurpleSoluble starch Yellow PurpleTrehalose Yellow PurpleSugar control Yellow PurpleNitrate Green-blue OrangeNitrate control OrangeCorn meal with Tween 80

with 79% showing positive assimilation in 2days and 97.5% in 3 days. The single isolate ofC. tropicalis that did not react was CBS 6418,which reportedly undergoes bud meiosis (12).This strain has an unknown requirement, as itdid not grow on any of the defined media.One hundred and eighty-seven isolates repre-

senting species less frequently associated withhumans were also examined with the Uni-Yeast-Tek kit. One hundred percent correlationwith conventional procedures was obtained;this correlation included 21 genera (includingBrettanomyces, Candida, Cryptococcus, Debar-yomyces, Endomycopsis, Geotrichum, Hansen-ula, Prototheca, Saccharomyces, Torulopsis,Trichosporon, Rhodotorula) and 48 species.Correct biochemical reactions were obtained,but insufficient data were provided with theUni-Yeast-Tek system for classification. Forcertain isolates, the use of the kit providedsufficient information for presumptive identifi-cation and, in the case ofPrototheca, definitiveidentification was possible. The emerging path-ogens Prototheca zopfi and P. wicherhamiigrew much better on the Uni-Yeast-Tek me-dium than in the broth. Geotrichum candidumcould be distinguished from Trichosporon capi-tatum and T. penicillatum with experience inrecognizing their distinctive morphologies onthe Uni-Yeast-Tek media.

DISCUSSIONThe selection of characteristics in the Uni-

Yeast-Tek kit were good choices for the identifi-cation of the common medically importantyeasts. The kit permitted the specific identifica-tion of C. albicans and C. stellatoidea mainlyon the basis of their production of germ tubes.The glucose-beef extract broth proved accepta-ble for germ tube production as compared withbovine serum or tissue culture medium 199.

VOL. 2, 1975 355

on May 30, 2018 by guest

http://jcm.asm

.org/D

ownloaded from

a rbL

CrdL

FIG. 1. Germ tube ofC. albicans without constriction at origin on yeast cell: (a) high-dry light microscopy,x750 (b) scanning electron microscopy, x6,500; elongated pseudohyphal cell ofC. tropicalis with constrictionat origin; (c) high-dry light microscopy, x750 (d) scanning electron microscopy, x5,285.

356

on May 30, 2018 by guest

http://jcm.asm

.org/D

ownloaded from

EVALUATION OF YEAST IDENTIFICATION SYSTEM

TABLE 2. Accuracy of the identification ofcommonclinical yeast isolates by the Uni-Yeast-Tek system

No. cor- % Correlation. ~~~~~~~with conven-Organism rect/no. wt ovn

tested tional identifi-cation

Candida albicans 85/85 100C. stellatoidea 9/9 100C.parapsilosis 46/46 100C. tropicalis 55/56 98.2C. pseudotropicalis 7/7 100C. guilliermondii 31/31 100C. krusei 29/29 100Cryptococcus albidus 22/22 100Cryptococcus laurentii 10/10 100Cryptococcus neoformans 35/35 100Cryptococcus terreus 3/3 100Rhodotorula rubra 29/29 100R. glutinis 4/4 100Saccharomyces cerevisiae 19/19 100Torulopsis glabrata 20/20 100Trichosporon cutaneum 31/31 100

Joshi et al. (6) previously showed that C. albi-cans produced germ tubes in various peptonebroths at 37 C. Of the three isolates of C. albi-cans that gave only sparse germ tube produc-tion in all preparations, two were from patientswith a history of chronic vaginitis and exten-sive polyene therapy. In general, fermentationof sugars and mycelial production by these twoisolates were weak. It has been observed thatexposure to polyene antibiotics alters the physi-ological and morphological characteristics of C.albicans (5). Our unpublished observations on

C. albicans from recalcitrant infections supportthis observation. The third isolate of C. albi-cans was obtained from the CBS as Syringo-spora albicans, the reported perfect stage of C.albicans. van der Walt (13) initially consideredthis strain a heterobasidiomycete but recentlyamended his description to include it in theascomycetes (14). The somewhat atypical mor-

phology and physiology of this strain are re-

flected in the confusion concerning its life cycleand taxonomy.Among the yeasts producing germ tubes was

the type culture of C. nouvelii CBS 6552. Saez(10) isolated this species from animals in a Pariszoo. C. nouvelii does not assimilate xylose andthus differs from the standard description of C.albicans. However, Ahearn (2) reported thatrare strains ofC. albicans from humans did notassimilate xylose. Therefore, we consider C.nouvelii a synonym of C. albicans.The identifications of most of the other clini-

cal yeast isolates by the Uni-Yeast-Tek kit,although proven correct in this study by a con-

ventional procedure, would have to be regarded

as presumptive. As with any limited system,verification of certain identifications requires amore complete assessment of morphologicaland physiological properties. Additional sugarassimilations (melibiose, dulcitol, inositol) andflorescent antibody tests or mouse pathogenic-ity tests are required to verify the identificationof Cryptococcus neoformans. C. parapsilosiscould not be distinguished from C. maltosa (7)due to the false-negative cellobiose reactions bythis latter species. Modification of the cellobiosemedium formulation to overcome the difficultyof interpretation of latent reactions would en-hance distinction of species of the C. tropicalisgroup. Fortunately, C. maltosa has not beenreported from clinical specimens and seems tooccur in nature mainly in oil-soaked soils. Theidentification of C. pseudotropicalis and C.guilliermondii also must be classified as pre-sumptive. For example, to differentiate be-tween C. pseudotropicalis and C. kefyr, theability to assimilate xylose must be deter-mined. Interest in identifying the above yeastsin clinical laboratories may increase in the fu-ture due to their potential as food yeasts. Bar-nett and Pankhurst (4) list C. guilliermondii,Debaryomyces hansenii, Pichia guilliermon-dii, and Torulopsis candida in a group withsimilar fermentation and assimilation charac-teristics. Combinations ofmorphological proper-ties, including ascosporulation characteristics,are necessary for their distinction. Species ofC.rugosa, C. valida, C. lipolytica, and the C.krusei complex also require further biochemicaltests, such as xylose, erythritol, D-mannitol,and sorbitol assimilation media, for definitiveidentification. Verification of Saccharomycescerevisiae would require the observation of asco-spores. Only three isolates of Cryptococcus uni-guttulatus and two isolates of Cryptococcus lu-teolus were examined. The isolates ofCryptococ-cus uniguttulatus, except for failure to utilizedulcitol, had the same assimilation pattern asmost of the isolates ofCryptococcus neoformans(erythritol-negative strains). Cryptococcus lu-teolus, which assimilates melibiose in contrastto Cryptococcus neoformans, and Cryptococcusuniguttulatus grow on most routine media attemperatures up to 33 to 34 C, whereas rarestrains of Cryptococcus neoformans fail to growin vitro at 37 C but do grow at 35 C. Althoughnot found in this study, the temperature rangesfor these species rarely may overlap if stricttemperature control is not maintained. There-fore, expanded sugar assimilation patternsshould be used for definitive identifications ofthe cryptococci.A major advantage of using the Uni-Yeast-

Tek system in clinical laboratories is that a

VOL. 2, 1975 357

on May 30, 2018 by guest

http://jcm.asm

.org/D

ownloaded from

358 BOWMAN AND AHEARN

single technician can inoculate 50 to 75 platesin 1 day. Most species can be identified pre-sumptively in 3 to 4 days; however, some maytake 6 days for complete results. The Uni-Yeast-Tek plates are easy to store and main-tain during growth, since they are incubated atroom temperature. One possible drawback ofthe kit is the need to keep it refrigerated priorto use. When the Uni-Yeast-Tek kit is assem-bled for commercial use, each plate will besealed with a plastic cover, making it air tight.This modification should increase the shelf lifebeyond the 2 to 3 months experienced in thisstudy.

In general, the Uni-Yeast-Tek kit shouldgreatly increase the capabilities of yeast identi-fication in routine clinical laboratories.

ACKNOWLEDGMENTSThis research was supported in part by the Microbiology

Research Fund of Georgia State University.We thank Corning Medical for their gift of materials

used in this study.

LITERATURE CITED1. Adams, E. D., Jr., and B. H. Cooper. 1974. Evaluation

of a modified Wicherham medium for identifying med-ically important yeasts. Am. J. Med. Tech. 40:377-388.

2. Ahearn, D. G. 1974. Identification and ecology of yeastsof medical importance, p. 129-145. In J. E. Prier andH. Friedman (ed.), Opportunistic pathogens. Univer-sity Park Press, Baltimore.

3. Ahearn, D. G., J. R. Jannach, and F. J. Roth, Jr. 1966.Speciation and densities of yeasts in human urine

specimens. Sabouraudia 5:110-119.4. Barnett, J. A., and R. J. Pankhurst. 1974. A new key to

the yeasts. American Elsevier Publishing Co., Inc.,New York.

5. Hamilton-Miller, J. M. T. 1972. Physiological proper-ties of mutagen-induced variants ofCandida albicansresistant to polyene antibiotics. J. Med. Microbiol.5:425-440.

6. Joshi, K. R., J. B. Gavin, and D. A. Bremner. 1973. Theformation of germ tubes by Candida albicans in var-ious peptone media. Sabouraudia 11:259-262.

7. Komagata, K., T. Nakase, and N. Katsuya. 1964. As-similation of hydrocarbons by yeast. II. Determina-tion of hydrocarbon-assimilating yeasts. J. Gen.Appl. Microbiol. 10:323-331.

8. Lodder, J. (ed.). 1970. The yeasts. North-Holland Pub-lishing Co., Amsterdam.

9. MacKenzie, D. W. R. 1964. Morphogenesis of Candidaalbicans in vivo. Sabouraudia 3:225-232.

10. Saez, H. 1973. Candida nouvelii, nouvelle espece delevure rencontree chez deua mammiferes sauvagesmorts en captivite. Bull. Soc. Mycol. Fr. 89:79-82.

11. Silva-Hutner, M., and B. H. Cooper. 1974. Medicallyimportant yeasts, p. 491-507. In E. H. Lennette, E.H. Spaulding, and J. P. Truant (ed.), Manual of clini-cal microbiology, 2nd ed. American Society for Micro-biology, Washington, D. C.

12. Sukroongreung, S., and L. Rodrigues de Miranda. 1973.A new aspect of the life-cycle of Candida tropicalis.Antonie van Leeuwenhoek J. Microbiol. Serol. 39:65-80.

13. van der Walt, J. P. 1970. The genus Syringospora Quin-quad emend. Mycopathol. Mycol. Appl. 40:231-243.

14. van der Walt, J. P., and E. Johannsen. 1973. The per-fect state of Torulopsis magnoliae. Antonie van Leeu-wenhoek J. Microbiol. Serol. 39:635-647.

15. Wickerham, L. J. 1951. Taxonomy of yeasts. U.S. De-partment of Agriculture Tech. Bull. 1029. U.S. De-partment of Agriculture, Washington, D.C.

J. CLIN. MICROBIOL.

on May 30, 2018 by guest

http://jcm.asm

.org/D

ownloaded from