Vol. 10, No. 3JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1979, p. 357-3640095-1137/79/09-0357/08$02.00/0

Evaluation of the New API 20C Strip for Yeast IdentificationAgainst a Conventional Method

G. A. LAND,t* B. A. HARRISON,' K. L. HULME,1 B. H. COOPER,2 AND J. C. BYRD3

Department ofMicrobiology, Wadley Institutes ofMolecular Medicine, Dallas, Texas 752351; Department ofMicrobiology, Baylor University Medical Center, Dallas, Texas 752462; and Department ofMicrobiology, St.

Paul Hospital, Dallas, Texas 752353

Received for publication 1 July 1979

The new API 20C yeast identification system together with appropriate micro-scopic morphology determinations achieved a 97% correlation with a rapid con-ventional method. Whereas a group composed of Candida, Torulopsis, Saccha-romyces, and Rhodotorula was identified with ease (98% overall correlation), asecond group, containing Cryptococcus, Trichosporon, and Geotrichum species,appeared to give the system the most difficulty (90% correlation). Within thisgroup particular difficulty was encountered in identifying varieties of Cryptococ-cus albidus, C. terreus, C. laurentii, Trichosporon beigelli, and Geotrichum spp.as to species. The API 20C system should be incubated the full 72 h prescribedby the manufacturer. However, when used in conjunction with appropriatemorphological tests, presumptive identifications of some Candida and Torulopsisspecies may be made at 24 to 48 h. To facilitate identifications of the moredifficult group of yeasts, ancillary tests for determining nitrate reductase, urease,and phenol oxidase activities should be considered as additions to the strip.Incorporating the phenol oxidase test would be especially important for identifi-cation of Cryptococcus neoformans, a yeast which should be identified as quicklyand as accurately as possible. The API 20C system with computer assistance hasproved to be an easy-to-inoculate, versatile, and fairly rapid method of yeastidentification, giving results comparable to those obtained by conventional meth-odologies.

The availability within the past several yearsof commercial products which aid in the acqui-sition and interpretation of data for identifica-tion of medically important yeasts has renderedthe task of obtaining this information much lessdemanding than it once was (7). The majority ofthe commercial products currently availableprovide carbohydrate assimilation tests in a con-venient plate or strip form. Some products in-corporate carbohydrate assimilation as well asother biochemical tests, and these productseliminate the necessity for preparing test mediaand simplify the storage of the large variety ofmedia required for identifying yeast isolates (15).Although based on traditional methodology, theminiaturization of biochemical tests in thesecommercial kits permits the reading of resultsafter a shorter period of incubation than wasfeasible with the earlier conventional methods(2, 13, 18, 19). Most manufacturers recognize thenecessity for conducting morphological exami-nations along with biochemical testing and rec-ommend such procedures in the instructions

t Present address: Division of Laboratory Medicine, Uni-versity of Cincinnati Medical Center, Cincinnati, OH 45267.

that accompany their products. As reported inrecent studies (4, 5, 7, 15, 20), it is now possiblewith the use of these commercial systems toreliably identify most medically importantyeasts within 48 to 72 h from the time biochem-ical tests are inoculated. In contrast, the moretraditional methods require a maximum of 14days for completion (1, 3, 9).The original API 20C yeast identification strip

(Analytab Products, Div. ofAyerst Laboratories,Plainview, N.Y.), which was one of the firstcommercial products to be introduced for testingmedically important yeasts, provided media indehydrated form for testing both carbohydratefermentation and carbohydrate assimilation.These properties, when used in combinationwith morphological characteristics, permittedidentification of many yeast species with a re-spectable level of reliability (5, 15, 20). However,certain aspects of the system were less conven-ient than was desired, and the data base whichhad been used to develop the system was limited,including only a few isolates of certain species ofyeasts commonly encountered in clinical labo-ratories. The main technical difficulty in using

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the system involved the complexities of fillingthe microtubules without trapping air bubblesin the agar, which subsequently led to misinter-pretation of fermentation tests (5). To circum-vent these relatively minor disadvantages and toimprove the overall capabilities of the system, a

new generation of the API 20C was introducedin early 1978. The new design incorporated thefollowing modifications of the original system:(i) the use of assimilation tests only; (ii) largercupules for easier filling; (iii) addition of new

substrates and elimination of those that hadbeen shown after extensive development evalu-ation to provide imprecise differentiations ofspecies based on computer-assisted interpreta-tion of test data (14); and, most importantly, (iv)clinical evaluation of the system with an exten-sive data base consisting of at least 20, and insome cases over 100, isolates of each of 16 yeasttaxa. With this new system, a binomial profilenumber can be derived from growth patterns on19 substrates. This profile number provides a

convenient method for comparing unknownyeast isolates with those numerical profiles inthe data base in order to derive an identificationof the unknown (14). For biotypes that are notfound in the profile index, a phone-in computerservice is available for determining a likely iden-tification of unusual isolates.The following is a report of an evaluation of

the revised API 20C, using clinical isolates de-rived from the diagnostic mycology laboratoriesof three hospitals along with stock isolates ofselected species. The purpose of the study was

to ascertain the level of reliability of the API20C system when compared with a rapid con-

ventional method (RCM) for yeast identification(11).

MATERIALS AND METHODS

Microorganisms. All of the yeasts used in thisstudy were either clinical laboratory isolates or stockcultures from the following sources: the Wadley Insti-tutes of Molecular Medicine, the Baylor UniversityMedical Center, and the St. Paul Hospital, Dallas,Tex., and the University of Oklahoma at Norman. Thefollowing American Type Culture Collection isolateswere also used: ATCC 26310, Candida albicans;ATCC 24064, Cryptococcus neoformans; ATCC 16725,Rhodotorula glutinis; ATCC 10663, Trichosporoncapitatum; and ATCC 9331, Trichosporon pullulans.Other isolates serving as known positive controls foridentification were proficiency testing samples fromthe New York City Department of Health, College ofAmerican Pathologists, Skokie, Ill., and center forDisease Control (Atlanta, Ga.) proficiency testing pro-grams. All clinical isolates were identified by one lab-oratory and then coded, randomized, and distributedto the other participating laboratories for identifica-tion and comparison. To complete the double-blind

J. CLIN. MICROBIOL.

study, all stock cultures and other strains serving aspositive controls were treated in the same manner.API 20C yeast identification system. The tip of

a wooden applicator stick was used to pick up a portionof a small colony of each unknown yeast to inoculatemolten (42°C) API 20C basal medium, and cupuleswere filled as per the manufacturer's directions. Alsoin accordance with the manufacturer's directions,yeasts were inoculated onto cornmeal agar plates viathe Dalmau culture technique for morphological de-terminations. Both morphology and assimilation testswere incubated at 30°C, and results were recorded inthe manner suggested by the API package insert.Upon the final observation of the API system, a profilenumber was assigned to each isolate and comparedwith profile numbers listed in the provided quickindex. Occasionally, one numerical profile was as-signed to two or even three species, in which case bothmorphology and ancillary biochemical tests (i.e., ni-trate reductase, fermentations, and urease production)suggested by API were used to determine the finalidentification. In those cases where an organism pro-duced a profile that was not listed in the API profileregister, the API supplementary computer identifica-tion system was utilized. Computer-assisted presump-tive identification was dependent upon the calculationof likelihood of occurrence between the unknownyeast's biochemical characteristics and isolates withsimilar characteristics present in the computer's databank.RCM. The RCM system of yeast identification, as

previously described (11), consisted of four biochemi-cal tests: a dye pour plate auxanogram, Tween-80-oxgall-caffeic acid (TOC) medium, a 10-min swabnitrate test, and a 4-h urease test with urea R broth(Difco Laboratories, Detroit, Mich.). Briefly, the testswere done as follows: a modified DPPA medium (10-12, 16), for determining yeast assimilation patterns on14 different carbohydrates, was composed of (per literof water): 20 g of agar, 0.67 g of yeast nitrogen base,and 20 mg of bromocresol purple. Ingredients werethen solubilized by heating, and the pH of the moltensolution was adjusted to 7.2 and sterilized. Sterile,molten dye agar medium was aseptically dispensed in60-ml portions into sterile prescription bottles andstored at 4°C until required. For assimilation testing,dye pour plate auxanogram medium in two bottles wasmelted, cooled to 40 to 43°C, inoculated with 5 ml ofa MacFarland no. 5 suspension of yeasts, poured intopetri plates (150 by 15 mm), and cooled. Individualstock solutions of 14 carbohydrates were made innormal saline (pH 7) at concentrations which woulddispense in 0.1 ml that amount of carbohydrate nec-essary for its optimum assimilation by yeasts. Stocksolutions were filter (0.45 [Lm; Millipore Corp., Bed-ford, Mass.) sterilized, and 0.1 ml of each was placedindividually on a sterile 0.5-inch (ca. 1.3-cm) concen-tration disk. The following carbohydrates were ar-ranged individually (7 per petri plate) on inoculatedand solidified dye medium as follows: plate 1, dextrose,galactose, sorbose, sucrose, maltose, cellobiose, andtrehalose; plate 2, lactose, melibiose, raffinose, mele-zitose, xylose, dulcitol, and inositol. Observations weremade after 24 and 48 h of incubation at 25'C, with apositive test recorded as either a reduction of the

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purple dye to yellow or, if the plate had completelyreduced to yellow, appearance of growth around thecarbohydrate disk.

Microscopic observation of pseudohyphae, hyphae,arthrospores, and blastospores were made on TOCplates. TOC medium has also been reported to pro-mote germ tubes and chlamydospores for appropriateCandida species, in addition to the above morpholog-ical characteristics (8). Caffeic acid added to the me-

dium served as a substrate for the phenol oxidasereaction, an enzymatic reaction presumably character-istic of C. neoformans (17). The TOC medium con-

tained 10 g of oxgall (Difco), 20 g of Davis agar, 0.3 gof caffeic acid, and 1 ml of Tween-80 brought to a boilin 1,000 ml of distilled water. The solubilized compo-nents were then autoclaved, and plates containing 30ml of medium were poured. Dried plates were streakedwith a sterile swab, depositing a heavy inoculum on

one corner of the plate and then continuing lightlywith the characteristic Dalmau technique, finally over-laying the lightest streak with a sterile cover slip. Theheaviest streak was used to rapidly detect the char-acteristic brown pigment of C. neoformans, whereasthe lighter streak was used to monitor specific mor-phological changes in the yeasts as they grew. Onceinoculated, plates were incubated at 37°C for 3 h, afterwhich they were inspected for germ tube productionand pigmentation, whereupon they were incubatedfurther at 25°C. The TOC plates were subsequentlyobserved at 6, 24, and 48 h for either brown pigmen-tation, chlamydospore formation, or other morpholog-ical changes.The presence of nitrate reductase in yeasts was

determined by swabs saturated with a fivefold-concen-trated liquid medium (pH 5.8 to 6.0) containing 2 g ofKNO3, 11.7 g of NaH2PO4, 1.14 g of Na2HPO4, and 1.2ml of a 17% solution of Zephiran chloride in 200 ml ofwater (11). Dried swabs were inoculated by sweepingthem across several colonies of a plate and then swirl-ing them against the bottom of an empty test tube (13by 150 mm) to ensure contact between organisms andsubstrate. The tube and swab were incubated for 10min at 45°C, and the swab was then placed in a secondtube containing two drops each of 0.5% ca-naphthyla-

API 20C STRIP EVALUATION 359

mine and 0.8% sulfanilic acid, each in 5 N acetic acid.A positive test was indicated by the swab tip turningbright cherry red.

For the urease test, one vial of urea R broth was

reconstituted with distilled water on the day it was tobe used. A 0.2-ml amount of the resultant liquid wasdispensed into each well of a 96-well microtiter testplate (Microtiter II; Falcon Plastics, Oxnard, Calif.). Aheavy inoculum (3 to 4 small colonies) of the unknownyeast was transferred via a wooden applicator tip tothe microtiter wells containing urea R broth. Wellswere sealed with clear sealing tape and incubated for4 h at 37°C. Observations were made hourly, with anychange of the straw-colored medium to pink consid-ered a positive test (11).

RESULTS

The overall results obtained with the new API20C correlated very closely with the RCM.There were 1,063 positive identifications out of1,093 total cultures (97%), using the API bio-chemical tests in conjunction with morphologyand a nitrate reductase test (Table 1). Ninety-two percent of these yeasts were compatiblewith profiles appearing in the system's profileindex; the remaining profiles (5%) were similarto profiles stored in the computer's data base.Using the biochemical tests alone, 75% of theisolates could be correctly identified with thecommercial system. The average time to positiv-ity for Candida, Torulopsis, Saccharomyces,and Rhodotorula species (group 1 yeasts) was31 h, as opposed to 72 h for a more difficultgroup (group 2), composed of Cryptococcus, Tri-chosporon, and Geotrichum species. C. albicansand Torulopsis glabrata could routinely beidentified within 24 h by using the API strip inconjunction with appropriate morphologicalchanges. Approximately 5% of the yeasts sur-

veyed required computer assistance for identifi-cation, and 3% were not identifiable by API 20C.

TABLE 1. Correlation ofAPI 20C with RCM in the identification of 1,093 yeasts from six medicallyimportant genera

Correlation' Avg time to positiveIdentification method No. of isolates

B B/M/NO3 API (h) RCM (h)

Quick indexGroup 1b 707 547 (77) 677 (96) 31 18Group 2C 386 275 (71) 337 (87) 72 30

Total 1,093 822 (75) 1,014 (92)

Computer aidedGroup 1b 21 (2.0)Group 2C 28 (2.6)

Overall correlation 1,093 822 (75) 1,063 (97)a The numerical and percent (in parentheses) correlations of API 20C with RCM by biochemical tests only

(B) or by biochemistry, morphology, and nitrate (B/M/NO3).b Isolates representing Candida, Torulopsis, Saccharomyces, and Rhodotorula species.c Isolates representing Cryptococcus, Trichosporon, and Geotrichum species.

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360 LAND ET AL.

In the clinical portion of this study, 98% ofgroup 1 yeasts were identified by the combina-tion of API, morphology, and nitrate reductase,whereas 86% ofthe organisms could be identifiedon the basis of assimilation results alone (Table2). One isolate of Candida solani and four ofeight isolates of Saccharomyces cerevisiae,whose profiles were not in the data base, wereparticularly difficult to identify with the APIsystem. Group 2 yeasts identified with the threecombined tests also had a 98% correlation ratewithRCM (Table 3). Morphology was extremely

TABLE 2. Clinical comparison of the API 20C yeastidentification system versus RCM: group 1(Candida, Torulopsis, Saccharomyces, and

Rhodotorula)

Organism No. of iso- Correlation'lates B B/M/NO3

C. albicans 64 89 100C. tropicalis 80 100 100C. parapsilosis 28 96 100C. krusei 15 0 100C. stellatoidea 3 100 100C. guilliermondii 3 0 100C. pseudotropicalis 3 100 100C. lipolytica 3 0 100C. solani 1 0 obT. glabrata 73 100 100T. candida 2 0 100S. cerevisiae 8 50 50bR. glutinis 3 100 100R. rubra 1 100 100

Avg 86 98

aPercent correlation of API 20C with RCM bybiochemical tests only (B) or by biochemistry, mor-phology, and nitrate (B/M/NO3).

b Some biotypes were not in the computer.

TABLE 3. Clinical comparison of the API 20C yeastidentification system versus RCM: group 2

(Cryptococcus, Trichosporon, and Geotrichum)

No. of iso-Correlation'

Organism lateslts B B/M/NO3

C. neoformans 26 85 100C. albidus var. al- 3 100 100

bidusC. albidus var. 3 100 100

diffluensC. laurentii 2 50 50T. beigelii 3 67 100T. capitatum 3 0 100G. candidum 1 100 100

Avg 74 98a Percent correlation of API 20C with RCM by

biochemical tests only (B) or by biochemistry, mor-

phology, and nitrate (B/M/NO3).

important in the identification of these orga-nisms, since only 74% could be determined bybiochemical tests alone. One clinical isolate ofCryptococcus laurentii could not be identifiedby both the combined tests and computer as-

sistance.Results obtained in the clinical study sug-

gested that the data obtained for the API stripwere often insufficient for separating Candidakrusei, Candida lipolytica, and T. capitatum as

well as in identifying Cryptococcus, Trichospo-ron, and Geotrichum species. The stock cultureportion of the study was weighted in favor ofthese organisms in order to provide a severe testof the capabilities of the API system. Amongthose yeasts in group 1 of the stock culturestudy, Candida stellatoidea proved to be themost difficult species for the API system toidentify consistently (Table 4). Approximately58% of the isolates utilized the API trehalose;however, according to the API percentage chart,only 1% should have reacted positively. TheRCM trehalose, on the other hand, was assimi-lated by all isolates of C. stellatoidea. Moreover,some 13% of these were negative on API maltose,whereas according to the percent chart andRCM all isolates should have grown on thesubstrate. These discrepancies gave the API sys-tem a 51% overall efficiency rating for the iden-tification of C. stellatoidea. Several organismswere found which had no correlating profile inthe API data base including: 4 of 15 isolates ofS. cerevisiae, 2 of 11 isolates of R. glutinis, 1 of2 isolates of R. rubra, 3 of 80 isolates of C.

TABLE 4. Comparison of identification of stockcultures by the API 20C system with RCM: group 1

(Candida, Torulopsis, Saccharomyces, andRhodotorula)

No. of Correlation'Organism ioaeisolates B B/M/NO3C. albicans 80 88.8 96.2C. parapsilosis 44 95.4 95.5C. tropicalis 80 100 100C. krusei 60 0 100C. stellatoidea 27 51.8 51.8C. guilliermondii 10 60 100C. pseudotropicalis 3 100 100C. lipolytica 8 0 100T. glabrata 73 100 100T. candida 2 0 100S. cerevisiae 15 46.6 74.0R. glutinis 11 27.2 73.0R. rubra 7 14.2 86.0

Avg 71 94

aPercent correlation of API 20C with RCM bybiochemical tests only (B) or by biochemistry, mor-phology, and nitrate (B/M/NO3).

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albicans, and 3 of 44 isolates of Candida par-apsilosis. These biochemically variable isolatesof C. albicans and C. parapsilosis also exhibitedaberrant morphologies, making their identifica-tions by either system difficult.

In addition, it was difficult to identify as tospecies stock cultures belonging to group 2 withthe API yeast system. Seventy-one percent ofthis group were identified on the basis of bio-chemical tests alone, and 86% were identified byusing the three combined tests (Table 5). Cryp-tococcus terreus and Geotrichum candidumwere the most difficult isolates to identify. Iso-lates of C. terreus varied in ability to assimilateinositol, with only 57% becoming positive after96 h of incubation. 2-Ketogluconate, a substrateutilized by the same yeasts that metabolize ino-sitol, also demonstrated the same variability inassimilation as did inositol, further compoundingthe problems in identifying these yeasts. Geotri-chum species did not regularly assimilate APIglycerol and xylose, making their identificationalso difficult. The problems experienced withthe API system in identifying other group 2organisms also related to false negative assimi-lations of inositol or other key substrates. Forexample, 3 out of 164 isolates of C. neoformans

API 20C STRIP EVALUATION 361

did not assimilate inositol after 96 h of incuba-tion, and 10 of 81 Cryptococcus albidus var.albidus and 5 of 26 C. laurentii (Table 6) alsofailed to assimilate this carbohydrate. Twelve of

TABLE 5. Comparison of the identification of stockcultures by the API 20C system with RCM: group 2

(Cryptococcus, Trichosporon, and Geotrichum)

No. of CorrelationaOrganism isolates B R/M/NO3

C. neoformans 138 93.1 94.3C. albidus var. al- 78 67.4 79

bidusC. albidus var. dif- 34 78.5 78.5fluens

C. laurentii 24 37.5 87.5C. terreus 7 28.5 28.5C. uniguttulatus 3 100 100T. beigelii 41 51.5 88T. capitatum 13 0 100T. penicillatum 2 100 100G. candidum 5 20 20

Avg 71 86

aPercent correlation of API 20C with RCM bybiochemical tests only (B) or by biochemistry, mor-phology, and nitrate (B/M/NO3).

TABLE 6. Comparison of selected positive assimilations in the API 20C system with RCMAssimilation (% positive)

Organism No. of isolates SubstrateaPredicted value API RCM

C. albicans 144 Mlz 2 13 0Gly 9 0 bAra 3 11 -

C. parapsilosis 72 Miz 99 100 100Gly 74 99 -Ara 99 94 -

C. tropicalis 160 Mlz 100 100 100Gly 11 35 -

Ara 3 7 -

Cel 12 8 86

C. stellatoidea 30 Tre 1 58 100

C. neoformansc 164 Ins 97 90 100Ara 8 14 -

Xlt 0 12 -

C. laurentiic 26 Ins 84 80 100Ara 99 95 -

Xlt 84 87 -

C. albidus var. albidusc 81 Ins 83 88 100Ara 88 80 -

Xlt 11 14a Mlz, Melezitose; Gly, glycerol; Ara, arabinose; Cel, cellobiose; Tre, trehalose; Ins, inositol; Xlt, xylitol.b_, Substrate not present in the RCM protocol.'A number of the cryptococci had to be incubated for an additional 24 h (i.e., 96-h total) in order for the

inositol reaction to become positive.

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TABLE 7. Yeasts identifiable by RCM which generated profile numbers not compatible with the API database

No. of CommentsOrganism iolate API profile no.Isolates

Candida humicoli 6 6777773 Same profile as C. laurentii, but different morphologyCandida solani 1 6102231Candida utilis 1 6404273Saccharomyces chevalieri 3 2040022 Same assimilation profile as S. cerevisiae with API,

but separable by RCMSaccharomyces champagni 1 2040032 Same as S. chevalieriKluveromyces fragilis 4 6660422 Same profile as Candida pseudotropicalis with API,

but ascospore positive and separable by RCMKluveromyces bulgaricus 3 6060422 Same as Kluveromyces fragilis

1 66624221 6460422

Kluveromyces lactis 1 6046673 Ascospore positivePichia ohmeri 4 6156372 Ascospore positiveRhodotorula 2 2670063Rhodotorula glutinis 1 6672062 Nitrate reductase positiveAureobasidium sp. 3 6777773 May at first be white and have same profile as C.

1 6773373 laurentii and T. beigelii, but becomes dematiaceouswith characteristic morphology upon aging

Ustilago sp. 1 2747573

81 isolates of C. albidus var. albidus, which were

lactose positive by RCM, were negative on thecorresponding API substrate and were for thatreason inseparable from their sibling species C.albidus var. diffluens.Of those organisms which were not identified

by the API 20C system, 50% were ascosporogen-ous yeasts (Table 7). Three isolates identified as

S. cerevisiae by API 20C were termed S. chev-alierii by the RCM. These organisms had simi-lar assimilation profiles but differed in theirfermentation patterns. Kluveromyces fragilis(four strains) and K. bulgaricus (three strains)had the same assimilation profiles as and similarmorphology to Candida pseudotropicalis, butthey differed in that they formed ascospores.

Candida humicola generated profiles similar tothose of both C. laurentii and Trichosporonbeigellii, so identification relied heavily upon a

critical evaluation of morphology, regardless ofthe system used. Another yeastlike organismwhich was confused with C. laurentii was Au-reobasidium species. These isolates appeared inearly culture as a white yeast with biochemicalproperties identical to C. laurentii or T. beigelii,but after extended culture they formed bothhyaline and dematiaceous hyphae.

DISCUSSIONThe new API 20C system had a high degree

of correlation with conventional methodology,providing that adjunctive tests of morphologyand nitrate reductase were used. The latter testwas especially important in identifying Crypto-coccus and Rhodotorula species. Pinello et al.

have placed special emphasis upon showing thatmorphological examinations in conjunction withthe API biochemical tests are necessary for com-plete yeast identification (14). This necessity formorphological examination in yeast identifica-tion was again underscored in our study by thefact that only 75% of these yeasts could beidentified on the basis of biochemical activityalone. To emphasize this point, the data havebeen presented both with and without morpho-logical characteristics being taken into consid-eration. The overall correlation of API withconventional methodology of 97% was in agree-ment with what Buesching et al. found (96%) inevaluating the system against 505 organisms (6).Paralleling our experience, Buesching et al. alsofound that fresh isolates appeared to grow morerapidly and to give fewer ambiguous reactionsthan did stock cultures.There are two clusters of medically important

yeasts which are of particular importance, and,for this reason, they must be rapidly and accu-rately identified. The first cluster consists of C.albicans, C. parapsilosis, and Candida tropi-calis, since it is possible for them to exhibitsimilar morphologies and assimilation patternson traditional media and substrates. The APIsystem is designed to separate members of thisgroup by their respective utilizations of melezi-tose, glycerol, and arabinose. Melezitose, accord-ing to the API data base, is assimilated by vir-tually all isolates of C. tropicalis and C. parap-silosis but by 2% of C. albicans isolates. Wefound that the degree of melezitose positivityamong C. albicans was much higher than the

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predicted percentage and would have led to anidentification of C. tropicalis or C. parapsilosis,especially for those isolates which did not formchlamydospores (Table 6). Glycerol, a substrateused for delineating C. parapsilosis from C.albicans and C. tropicalis, yielded similar incon-sistencies. Glycerol was assimilated by virtuallyall C. parapsilosis isolates, but not by C. albi-cans, whereas over one-third of the C. tropicalisisolates were positive, instead of the expected11%. It appeared that arabinose assimilation wasnot as variable a characteristic as those aboveand served as an excellent means of separatingC. parapsilosis from C. albicans and C. tropi-calis.The addition of a germ tube test to API 20C

would provide a good backup test for cornmeal-Tween-80 agar morphology and would also helpto split C. albicans away from C. parapsilosisand C. tropicalis (7, 17). This additional mor-phological test in tandem with melezitose, glyc-erol, and arabinose assimilations would help toremove some of the ambiguity in relying uponassimilation entirely as a means of identification.Cellobiose assimilation has been shown to be anefficient means of separating C. tropicalis fromC. albicans and C. parapsilosis (4-6, 11), and itcould also augment the other key API substratesif the problem of its variable assimilation couldbe overcome. The assimilation of cellobiose aswell as other carbohydrates by yeasts has beenshown to be a function ofa specific concentrationrange for each substrate and of the nitrogencontent of the medium (12).The failure to utilize the optimum concentra-

tion for each substrate as well as the relativelyhigh nitrogen content of the medium might alsoexplain the failure of several other yeasts togrow on substrates that should have been assim-ilated. Trehalose, commonly metabolized by C.stellatoidea in other systems and used by all ofthese isolates on RCM, had a 50% false negativerate on API. Furthermore, inositol, a key sugarin the conventional identification of Cryptococ-cus species (3, 11, 13, 17), also was not utilizedby 100% of cryptococci grown on API medium.Particularly noteworthy was the fact that only26% of C. terreus isolates utilized this carbohy-drate. Among the cryptococci, inositol assimila-tions were so weak that they had to be held anextra day to be considered positive, and 10 to15% didn't grow at all. 2-Ketogluconate, abackup test for inositol assimilation, had a sim-ilar 10 to 15% false negative rate instead of thenear 100% positive rate expected from the per-centage chart. These false negative assiimilationsled to some difficulty in identifying the variouscryptococci, an experience also noted by Buesch-

API 20C STRIP EVALUATION 363

ing et al. (6).The second cluster of yeasts of medical inter-

est are composed of C. neoformans, C. laurentii,and C. albidus. These species appear to be fairlyseparable by the API system. Xylitol appears tobe an adequate substrate for delineating C. lau-rentii from C. neoformans and C. albidus, with100% of C. laurentii utilizing the substratewhereas only about 14% of the other cryptococciwere positive. Arabinose was assimilated by 12%of the C. neoformans isolates in this study ratherthan the predicted 0%. This would still be a faircharacteristic for identification, since 95% of theC. laurentii and 80% of the C. albidus metabo-lized the substrate. However, due to the medicalimportance of determining the presence of C.neoformans in a clinical specimen, we feel anadjunctive test for phenol oxidase activityshould be provided with the kit. This test ispresumably specific for the identification of C.neoformans and could easily be adapted to theAPI 20C (8, 11, 17).Based upon our experience with the new API

20C yeast identification system, we conclude thefollowing: this identification system, togetherwith the recommended morphological tests, cor-relates well with conventional methodology.However, some of the biochemical tests chosenby the computer as a means of separating cer-tain taxa and the computer's use of these datadiffer considerably from conventional tests anddichotomies but may, with time, prove equal toconventional methods or perhaps even to be amore accurate approach to the identification ofmedically important yeasts.

ACNOWLEDGMENTThis work was supported in part by the Sammons Foun-

dation, Dallas, Tex.

LITERATURE CITED

1. Adams, E. D., Jr., and B. H. Cooper. 1974. Evaluationof a modified Wickerham medium for identifying med-ically important yeasts. Am. J. Med. Technol. 40:377-388.

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3. Ahearn, D. G. 1974. Identification and ecology of yeastsof medical importance, p. 129-146. In J. E. Prier and H.Friedman (ed.), Opportunistic pathogens. UniversityPark Press, Baltimore.

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