Rapid Methods for Identification of YeastsM. HUPPERT,* GLORIA HARPER,' SUNG H. SUN, AND V. DELANEROLLE
Mycology Research Department, Veterans Administration Hospital, Long Beach, California 90801
Received for publication 10 April 1975
Opportunistic infections by yeasts have been implicated as one of the majorcauses of complications in the compromised patient. Rapid recognition andidentification of these yeasts is essential for patient management, but conven-tional liquid medium methods for completing identification tests are cumber-some and time consuming. Rapid tests have been devised based on modificationsof methods commonly used in bacteriology. These rapid methods included testsfor carbohydrate and nitrate assimilation, fermentation, and urease production.These were compared with several current methods for accuracy of results, fortime to final identification, and for economy of time and reagents. In addition,the usual tests for pseudogerm tube formation, for production of hyphae orpseudohyphae, and for growth temperatures were included. The rapid testsachieved 96% or better accuracy compared with expected results, and 46 speciesof yeasts were identified in 1 to 2 days compared with the 10 to 14 days requiredby conventional liquid culture methods.
Modern medicine, with the capacity for pro-longing the life of a patient, has seen a markedincrease in secondary infections among pa-tients whose resistance has beeen compromisedby debilitating chronic disease, by therapeuticmodalities, and by severe injury. Fungi are acommon cause of infections in such patients (7,9, 11, 12, 14, 16, 19, 24). This increased fre-quency in the mycoses has been notable in twoadditional respects: greater severity of the dis-ease and greater diversity in etiological agents(2, 8, 10, 16, 21, 22, 25). Yeasts are among themost common etiological agents.
Since many species of these yeasts had beenconsidered innocuous previously, their recoveryfrom a pathological specimen presents prob-lems for the laboratory which must identify avariety of isolates and for clinicians who mustjudge their significance. Conventional liquidculture methods for speciation of yeasts arecumbersome and time consuming (15, 25, 27,28). Rapid methods have been proposed but re-strictions in the number of tests used limitsidentification to relatively few species (1, 5, 6,8). In addition, most clinical laboratories lackpersonnel with more than minimal trainingand experience in identifying fungi.The present study has been limited to identifi-
cation of yeasts in the genera Candida, Crypto-coccus, Torulopsis, and Trichosporon. Geotri-chum candidum, although not a yeast, hasbeen included because it occurs frequently with
'Present address: Clinical Laboratory, Little Companyof Mary Hospital, Torrance, Calif. 90503.
yeasts but can be differentiated from the latterby the same procedures. Two objectives wereplanned. The first involved the developmentand evaluation of rapid methods for identifyingalmost all species of yeasts recovered from hu-mans (15). The second required that the meth-ods should be familiar to technologists compe-tent with only bacteriological techniques. Sincemany of these personnel lack experience in my-cology, the tests employed are almost all physio-logical, and morphological criteria have beenlimited primarily to observation of whether hy-phae (or pseudohyphae) are present or absent.A most essential first step for speciating yeastsis recognition of whether or not perfect stageforms are present, e.g., asci and ascospores.Descriptions of these are beyond the scope ofthe present study but are available in referencetexts (8, 13, 15, 25), or such cultures can bereferred to a zymologist for identification.When no information about sexual spore formsis available, all yeast identifications must beconsidered presumptive.
(This paper includes, in part, a thesis submit-ted by G. H. to the California State University,Long Beach, in fulfillment of the requirementsfor the Master of Science degree.)
MATERIALS AND METHODS
The procedures described in this section representthe optimal methods derived from this study. Varia-tions which were studied are presented in later sec-tions.
cies (Table 1) were maintained as stock cultures onyeast-malt agar (8, 15).
Preparation of inoculum. Dense suspensionswere prepared from confluent growth at room tem-perature (24 to 48 h) using 6 ml of sterile water perpetri dish (100 by 15 mm) followed by transferringthe suspension to a sterile tube (16-mm diameter).Although quantitative standardization was per-formed by optical density (OD) determination and
by cell count, a practical and satisfactory estimate ofcell density was obtained when the suspension com-pletely obscured the black lines on a standard Wick-erham card (3 India ink lines approximately 0.75-mm wide on a white card). The less dense inoculumrequired for some of the conventional methods em-ployed for comparison with the rapid methods wasachieved by diluting the dense inoculum until theblack lines were visible as dark bands.
Carbohydrate assimilation. The following carbo-hydrates were used: glucose, maltose, sucrose, inosi-tol, lactose, cellobiose, raffinose, melibiose, erythri-tol, xylose, galactitol (dulcitol), and trehalose.These were selected on the basis of critical value fordifferentiating among species (15). The purest gradereagents available were used. For our modified aux-anographic method, dry disks containing carbohy-drate plus nutrient were prepared. Others have useddisks saturated with carbohydrate only (1, 5, 25). AlOx concentration (8.04 g/120 ml of distilled water)of commercially available yeast nitrogen base(YNB) (Difco Laboratories, Detroit, Mich.) was ad-justed to pH 6.40 ± 0.05 with 10% NaOH. A 20%solution of each of the 12 carbohydrates was pre-pared by adding 2 g of carbohydrate to 10 ml of thelOx concentrate of YNB and dissolving with mildheating. Solutions were sterilized by passagethrough a membrane filter (0.20 ,um) followed byaseptic transfer to sterile vaccine vials. A layer ofsterile blank 0.25-inch (ca. 0.64-cm) disks (concentra-tion disks, Difco) in petri dishes was saturated withone of the carbohydrate solutions by dropwise addi-tion from a syringe and needle. These were dried 48to 72 h in an evacuated desiccator containing anhy-drous CaSO4. The disks containing inositol, raffi-nose, and maltose required a second saturation afterthe first drying period.
The basal agar for the carbohydrate assimilationtest contained agar (20 g/1,000 ml), bromothymolblue (0.16 g/1,000 ml), and phosphate buffer (100ml/1,000 ml). The phosphate buffer stock solutionwas a 1:1 mixture of 0.067 M Na2HPO4 and 0.067 MKH2PO4 (pH 6.8). The melted agar was dispensedwhile hot into 2-oz. (ca. 0.06-liter) screw-cap bottles(approximately 30 ml/bottle). Volumes of 30 to 50 mlcan be used, but the smaller volume enhanced thefinal reading of growth because of the thinner layerof agar. The bottles of agar were autoclaved for 15min at 121 C. If tightly capped after cooling, thesebottles can be stored indefinitely at room tempera-ture.
The assimilation test was performed by pouringone bottle of melted agar for each strain to be testedinto sterile petri dishes (150 by 15 mm). Afterhardening, the plates were streaked for confluentgrowth with a swab saturated in the dense suspen-sion of cells. The streaked plates were allowed to drybefore applying the disks, usually 30 min. Diskswere applied to the surface of the agar using atemplate (Fig. 1) under the dish to insure placingthe disks equidistant apart. The position of the glu-cose disk was marked on the bottom of the petridish. Plates were incubated at 25 C (room tempera-ture) and read daily for 3 days. The earliest indica-tion of a positive reaction was a change of the indica-
'ATCC, American Type Culture Collection; CBS,Centralbureau voor Schimmelcultures; BNCYC,British National Collection of Yeast Cultures.
tor color from blue-green to yellow surrounding thedisk. This could be confirmed later during the incu-bation period by enhanced growth of the organism inthe area surrounding the disk. Confirmation byreading for growth may be required with those orga-nisms which produce acid from many carbohydrates,turning large sections of the plate completely yel-low. Under these circumstances, if no enhancedgrowth is evident around individual disks, the testis negative for that carbohydrate. This procedurewill be referred to as the YNB method in subsequentsections.The results obtained by the YNB method were
compared with those obtained from three additionalmethods. The Wickerham liquid medium methodwas performed without agitation according to theprocedure described in the Lodder text (15). Theoxidation-fermentation (OF) tube method followedthe procedures described by Webb et al. (27), exceptthat commercially available OF media (Difco) wasused. Tubes in both methods received 0.1 ml of di-luted inoculum. The last method used OF media in apetri dish. These were streaked heavily as in theYNB method, and dry disks containing only carbohy-drates were placed on the surface. Concentrations ofcarbohydrates were identical to those used in theYNB method. For the comparative studies, all testswere incubated at room temperature and read atintervals over a 14-day period.
Nitrate assimilation. Dry disks containing asource of nitrogen plus nutrient were prepared in amanner similar to that used for carbohydrate assimi-lation. A 1% KNO3 solution in a 4 x concentration ofyeast carbon base (Difco) (15, 25, 28) was used toimpregnate the disks. Disks for use as a positivecontrol were saturated with a solution of 1%(NH4)2SO4 in a 4 x concentration of yeast carbonbase. Disks for negative controls or persisting endog-enous nitrogen source contained only 4 x concentra-tion of yeast carbon base. The methods for steriliz-ing solutions and for saturating and drying the diskswere the same as described for carbohydrate assimi-lation. The basal agar for nitrate assimilation con-sisted of agar (20 g/1,000 ml) with bromothymol blue(0.08 g/1,000 ml). No phosphate buffer was added tothis medium, and adjustment to a light green color(approximately pH 6.8) may be required. The me-dium was dispensed in approximately 20-ml/bottleamounts and autoclaved for 15 min at 121 C.
Testing for assimilation of nitrate was requiredfor every species according to the scheme developed.One bottle for each strain was melted and pouredinto sterile petri dishes (100 by 15 mm). Plates werestreaked with dense inoculum and dried, and appro-priate disks were added in the manner described forcarbohydrate assimilation. Incubation was at 25 Cfor 24 to 48 h. A positive nitrate assimilation testwas indicated by a color change from light green todark blue surrounding the disk containing KNO:1.The color change was caused by the residual,strongly alkaline K+ after reduction of the NO:r-. Acolor change to bright yellow developed around the(NH4)2SO4 disk, resulting from utilization of theNH4, and an acid reaction from residual SO4= com-
FIG. 1. Template placed under petri dish for spot-ting disks and reading results. Abbreviations forcarbohydrates (clockwise for outer and inner circles)correspond to sequence in Table 3 beginning withglucose.
bined with acid from oxidation of the glucose in theyeast carbon base.
The conventional methods used for comparisonwere the Delft auxanographic plate (27) and theWickerham liquid medium procedures (8, 27). Di-luted inoculum (0.1 ml) was used, and incubation forboth was at room temperature. The Delft plate testwas read at 48 and 96 h, and the Wickerham test wasread daily for 7 days. A positive by the latter methodrequired subculturing to a second tube to determinewhether growth in the first tube could have beencaused by carryover of a source of nitrogen.
Fermentation tests. One milliliter of the heavysuspension was placed in each ofthree 1 1-mm-diame-ter test tubes. (The tubes need not be sterile.) Glu-cose, maltose, and sucrose fermentation tablets(Key Scientific Products, Los Angeles, Calif.) wereadded one to a tube. One milliliter of molten vasparwas added to each tube carefully with a Pasteurpipette so as not to trap any air bubbles. The vasparwas a mixture (1:1, wt/wt) of petrolatum jelly andparaffin (melting point, 48 to 56 C). The tubes wereincubated at 37 C for 24 h. A positive fermentationreaction was indicated by a rise of the vaspar plug,which separated completely from the liquid suspen-sion because of gas production.
Results with this rapid fermentation tests werecompared with those obtained by the conventionalDurham inverted tube procedure (8, 27). Tubes re-ceived 0.1 ml of diluted inoculum and were incu-bated at room temperature with final readings at 14days.
Urease production. One milliliter of the heavysuspension was placed in an 11-mm-diameter testtube (nonsterile). One urease tablet (Key ScientificProducts) was added, and the tube was incubated at
37 C for 24 h. A bright pink color at the end of theincubation period denoted a positive reaction.
The conventional method used for comparisonwas Christiansen urease agar slants (8, 27). Slantswere inoculated with 0.1 ml of the dense suspension,incubated at room temperature, and read daily for 5days.
Pseudogerm tube test. Most investigators referto this as the germ tube test. Mackenzie (17, 18) hasstated that the term germ tube ". is a misleadingone, as the similarity to the true germinative proc-ess of fungal spores is both superficial and tran-sient." He proposed the descriptive term "pseudo-germ tubes" for these structures characterized by". the production (often multiple) from any part ofthe surface of the parent cell and absence of proxi-mal constrictions. ." (17). All strains were studiedfor pseudogerm tube formation by adding 1 drop ofthe dense inoculum to 0.5 ml of fetal bovine serum ina test tube (10 by 75 mm) (8). The tubes were incu-bated at 37 C and read at 3 h for the formation ofpseudogerm tubes.
Production of hyphae or pseudohyphae. Thepresence or absence of hyphae or pseudohyphae wasdetermined in corn meal agar (without glucose) con-taining 1% Tween 80 (CMT) in a petri dish (50 by 12mm). The inoculum was obtained from the harvest-ing plate prior to preparing the dense suspension. Asmall amount of growth on a stiff needle was pressedthrough the agar to the bottom of the plate and thendrawn through the agar at an angle to make a longcut. Incubation was at room temperature for 24 to 48h. Production of hyphae was observed by invertingthe plate on the stage of the microscope and viewingwith the low-power (10x) objective lens.
Additional tests. For some species, temperaturetolerance (i.e., growth at 20 and 37 C) was requiredfor identification. The 20 C tolerance was observedusing the yeast-malt agar plate prior to harvesting.An additional plate of yeast-malt agar was incu-bated at 37 C when this information was needed forfinal identification. If morphology was required todifferentiate among several species, microscopicexamination was made on the surface of the CMTplate.
RESULTSDensity of suspension. Since rapid results
with these new procedures required a heavyinoculum, it was necessary to determine theupper and lower limits of inoculum density forobtaining accurate and reproducible results.The wavelength (Coleman junior spectropho-tometer, model 6D) yielding maximum sensitiv-ity for detecting a difference in density of cellswas determined using two dilutions of selectedspecies from the genera Candida, Cryptococ-cus, Torulopsis, and Trichosporon. The ODsfor these dilutions were read at several wave-lengths in a scan of the visible spectrum. Thewavelength yielding the greatest difference be-tween the two curves was chosen as the oneproviding maximum sensitivity for these yeast
cell suspensions. A typical example appears inFig. 2. The wavelengths of maximum sensitiv-ity for all species clustered around 580 nm.Upper limit density studies were performed byharvesting successive plates of the several spe-cies with the initial 6 ml of water until thesuspension attained a viscosity precluding theharvesting of additional plates. This and sev-eral serial dilutions were used to perform thecarbohycirate and nitrate assimilation, ureaseproduction, and fermentation tests. Accurate,reproducible, and rapid results were obtainedwith all suspensions yielding an OD - 0.6 (ap-proximately 0l cells/ml), and it was concludedthat only a minimum limit for inoculum den-sity was required. A suspension in a 16-mm-diameter tube which obscures the lines on aWickerham card has an OD - 0.6, and thissimple card method was used routinely.Carbohydrate assimilation. The four meth-
ods were evaluated in terms of percentage ofagreement with expected reactions for each spe-cies as published in Lodder (15), referred to asthe "reference." The combined results for all 46species with each of the four methods are pre-
01
0 8-
0 0 6-F-1
06LL
-j
00Q4-
0L0
0.2-
450 500 550 600 650 700(580)
WAVELENGTH (NM)FIG. 2. Typical example for determining the wave-
length which produced the maximum sensitivity fordetecting differences in density of cells in a suspen-sion. Species was C. guilliermondii.
sented in Table 2 and Fig. 3. The rapid YNBmethod produced 90% of the expected resultswithin 24 h and 97% agreement with the refer-ence by 48 h. The Wickerham method requiredat least 3 to 5 days to attain the same level ofagreement as the YNB procedure, although theformer achieved 100% agreement by 14 days.This was expected since the results published inthe reference were obtained with the Wicker-ham method. The two procedures using OFmedia were not as good. The OF plate methodwas particularly unsatisfactory because colorchanges began to revert on day 3 from yellowback to blue as the carbohydrate was ex-hausted, peptone was utilized as a carbonsource, and the medium became alkaline.Adams and Cooper (1) reported similar difficul-ties with OF media. The data in Table 2 and
TABLE 2. Comparison of results obtained by fourmethods for assimilation of carbohydrates
"Results are presented as percentage of agree-ment with reference (15) -+- standard deviation.
COMPOSITE COMPARISON OF CARBOHYDRATE
l00o, ASSIMILATION TESTS
90_ 1
80_
70-
60
3 - 5TYIME -DAYS
FIG. 3. Comparison of composite results obtainedby four methods for assimilation of carbohydratesexpressed as percentage of agreement, with expectedresults as published in reference 15. Symbols forbars: dotted, OF plate method; cross-hatched, OFtube method; striped, YNB rapid method; solid,Wickerham method.
Fig. 3 do not reflect this reversion for the 3- to 5-day and 6- to 14-day periods, but only that amaximum of 86% agreement had been obtaina-ble by this method. In preliminary studies YNBhad been used in the medium in plates ratherthan in the disks which contained only thecarbohydrates. The same reversion of indi-cator color occurred, and this was discarded infavor of incorporating nutrient and carbohy-drate in disks. In the latter method, reversionof indicator color did not occur (Fig. 4).The YNB method was less variable during
the first 2 days than the other methods (Table2). The principal cause for this variability isillustrated in Fig. 5-7 for the three carbohy-drates trehalose, cellobiose, and raffinose. Thepercentage of agreement with the referenceafter 24 h is relatively low for all three of thesecarbohydrates by each of the four methods. By48 h, however, the YNB method achieved 96%or better agreement with these three carbohy-drates, whereas the other methods attainedonly 82% at best by this time. These resultsindicate that factors which presented problemsfor the conventional liquid media methods hadrelatively little effect on the rapid YNB methodfor carbohydrate assimilation.
Fermentation tests. The rapid method andconventional Durham tube fermentation testswere evaluated in terms of the published ex-pected results (15) for each species. The rapidtest (Fig. 8) produced accurate results in 1 daywith 94% or better of the expected reactions,whereas the conventional Durham tube testrequired 14 days for comparable agreement.Two-thirds of the 6% incidence of nonagree-ment using glucose in the rapid test were withspecies which yielded positive reactions when anegative test was expected. This may have re-sulted from incubation at 37 C with the rapidprocedure, since the reactions reported in thereference were based on incubation at 25 to 28 Cfor 24 days. In addition to yielding resultsquickly, the rapid test was very easy to read.When fermentation had occurred, the vasparplug had been raised several centimeters inalmost every instance.
Nitrate assimilation. A comparison of re-sults obtained by the rapid method and by theDelft and Wickerham procedures indicated 100%agreement with the reference by all three meth-ods. Incubation time for the YNB method wassignificantly shorter, 1 to 2 days versus 4 to 14days. The change in indicator color from lightgreen to dark blue around the KNO3 disk wasobvious and dramatic with all species assimilat-ing nitrate, but either no change or a slightyellowing occurred with species which were neg-ative for nitrate assimilation. The disk contain-
Typical example of carbohydrate assimilation results by the YNB rapid method (C. guilliermondii
Pseudogerm tube production. As expected,C. albicans and C. stellatoidea produced pseu-dogerm tubes consistently within the 3-h incu-bation period. However, C. tropicalis, C. parap-silosis, and Cryptococcus gastricus also pro-duced structures which resembled pseudogermtubes. Although these were elongated blasto-spores, personnel inexperienced with the mor-phology of yeasts might not recognize the differ-ence and an incorrect identification could re-
100,
90 _
TIME DAYS
FIG. 5. Results obtained by four methods for as-
similation of trehalose. (See Fig. 3 for key to bargraphs.)
ing (NH4),S04 was surrounded by a yellow zone
with all species, and the area around diskscontaining yeast carbon base without a sourceof nitrogen either remained light green or
showed a slight change to yellow. Therefore,carryover of endogenous nitrogen did not influ-ence results with the rapid test, and there was
no need for starving cells or subculturing. Infact, the medium control disk was not required.
<
Is
70
60 _
50_
40
1 2 3 -5 6-14
TIME DAYS
FIG. 6. Results obtained by four methods for as-
similation of cellobiose. (See Fig. 3 for key to bargraph.)
sult. In our opinion complete reliancpseudogerm tube test for identifyingcans or C. stellatoidea by inexperiencenel may not be justified (see DiscussilHyphae or pseudohyphae forma
species in the genera Cryptococcus andsis failed to form any structures rehyphal formation in the CMT agar. Inall Candida and Trichosporon specieEstrain of G. candidum were positive. Inize that occasional strains of C.mondii and C. parapsilosis may not
100 _.
90+_
80_ t
RAFFINOSE
IS
"I
i
2 3TIME -DAYS
FIG. 7. Results obtained by four methosimilation of raffinose. (See Fig. 3 for kgraphs.)
ce on the obvious hyphal structures, and this has beenC. albi- considered in the construction of our schemad person- for identification of these yeasts (see below).On). Urease production. A comparison of resultsLtion. All obtained by the rapid method and by the con-Torulop- ventional method showed 100% agreement withsembling the reference by both methods. The principalcontrast, advantage of the rapid method was the muchs and the shorter incubation time, i.e., 1 day versus 5We recog- days.guillier- Quality controls for assimilation, urease,produce and fermentation tests. Satisfactory quality
control for these tests required a suspensionyielding positive reactions and one resulting innegative tests. No single species produced posi-tive results in all tests. Several strains in combi-nation were tried, and the best results wereobtained with C. pelliculosa and Cryptococcuslaurentii. Each species was grown and har-vested separately and then tested for satisfac-tory inoculum density. Equal aliquots of eachsuspension were mixed, and the mixture wasused to perform the rapid procedures. All therapid tests were definitely positive within 48 h.If preferred, suspensions of these two speciescould be used separately rather than mixed intoa single suspension. Our strain of Cryptococcuslaurentii assimilated all 12 carbohydrates andwas urease positive. The C. pelliculosa strainproduced positive reactions in tests for nitrateassimilation and fermentation. A suspension of
Eds for as- Trichosporon capitatum was used as a negativeiey to bar control. Our strain of this species assimilated
only glucose and was negative in all other tests.
GLUCOSE100I
90~~~~~~~~I
3..80-
LU
MALTOSE SUCROSE
TIME - DAYS
FIG. 8. Comparison of results obtained by the rapid and by the Durham tube fermentation tests expressedas percentage of agreement, with expected results as published in reference 15. Symbols: striped bar, rapidmethod; solid bar, Durham tube method.
Schema for identification of yeasts. Thisseries of tests would not be complete withoutpresenting a schema by which the results couldbe used easily for identification of these imper-fect yeasts. A schema has been constructed forthis purpose (Table 3). It includes all species inthe genera Candida, Cryptococcus, Rhodoto-rula, Torulopsis, and Trichosporon which, ac-cording to Lodder (15), have been recoveredfrom humans or animals. In addition, Saccharo-myces cerevisiae and G. candidum (not a yeast)have been included because ofthe need to distin-guish these common fungi from species in theabove five genera.The principal objective guiding the construc-
tion of this schema was to include only thosereactions which were most important for differ-entiating among species, keeping in mind thatmany personnel in clinical laboratories havelittle experience with these organisms. A pri-mary separation was made on the basis ofwhether hyphae or pseudohyphae were eitherwell developed or absent (or rudimentary) asseen in the CMT agar. The two species of Can-dida (C. guilliermondii and C. parapsilosis),in which occasional strains fail to produce pseu-dohyphae, appear under both categories. Simi-larly, occasional strains of R. glutinis and R.rubra may produce pseudohyphae, and theseappear in both sections of the schema. Specieswith variable reactions in some tests appearseveral times, when the result with a singlereagent might be positive and again when itwould be negative. Therefore, decisions in al-most all cases were reduced to simply positiveor negative results. Groups of species wereformed on the basis of assimilation tests fornitrate, glucose, maltose, and sucrose. Thesegroups could be differentiated into species byreading results for only those additional testswhich were critical within the group. Prelimi-nary trials with this schema by technologistsunfamiliar with the yeasts have met withgeneral acceptance and have resulted in suc-cessful species identifications of yeasts previ-ously reported incompletely, e.g., unidentifiedyeasts or Candida species that are not C.albicans. An additional benefit is gained byincluding only critical reactions in the schema.Almost all the reactions responsible for thelack of agreement between the rapid methodsand the reference have been eliminated.
DISCUSSIONAchievements by modern medical science
have produced a population of people uniquelysusceptible to infection by microorganisms pre-viously considered innocuous. Some of these
organisms exist saprophytically in man's envi-ronment, whereas others occur comensally, andthe individual with compromised defenses can-not escape eventual exposure. The yeasts, as agroup, are involved frequently in these oppor-tunistic infections.The true incidence of secondary infections
caused by yeasts is difficult to assess since,undoubtedly, many of these have not been rec-ognized. Quie and Chilgren (23) have statedthat "only 30% of disseminated candida infec-tions are diagnosed correctly antemortem sothat improved methods of diagnosis are clearlyneeded." There is ample evidence, however,that not only morbidity but also mortality mustbe significant. Law, MacMillan, and their asso-ciates (14, 19) reported studies on 427 burn pa-tients. Candida species were recovered one ormore times from 63.5% of the patients. In addi-tion to positive cultures from the wound, Can-dida species were found in urine (48%) and inblood (5%), and, of the 65 patients who died, 14(22%) deaths were attributable to disseminatedcandidiasis. Miller et al. (20) reported that inthe Center for the Study of Trauma at theUniversity of Maryland 16% of the nosocomialinfections were caused by Candida species, ex-ceeded only by Pseudomonas infections (24%).These were primarily shock trauma and postop-erative thoracic surgery patients, with only aninsignificant number of burn cases. Gaines andRemington (7) reviewed surgical records for the1960-1967 period and found 42 cases developingsystemic candidiasis, with 19 deaths. Theypointed out that candidiasis was not diagnosedearly enough for appropriate treatment in morethan 50%. Rifkind et al. (24) described 107 casesreceiving renal transplants, with 51 deaths. Sys-temic mycoses were involved in 23 (45%), Can-dida species accounting for 14. Hutter and Col-lins (9) found 202 mycoses in cancer patients,with yeasts responsible for 130. Eighty-four ofthe latter occurred in the leukemia-lymphomagroup. Kahanpaa (10), in an extensive surveyinvolving 8,290 specimens, identified a total of35 species of yeasts in 21% of bronchial secre-tions, 34% of lung tissue specimens, 71% ofautopsy lung specimens, and 20% of the pleuraleffusions. One must conclude that yeasts repre-sent a significant problem in these diseases andthat early recognition is essential to insure ap-propriate therapy.Our approach to the development of methods
for identifying yeasts has recognized these re-quirements. The procedures are adapted almostentirely from those familiar to microbiologistswith little experience in mycology, and the re-sults are obtained rapidly, usually in 2 to 4 days
after primary isolation compared to 10 to 14days by conventional liquid culture methods. Inaddition, the presumptive identificationscheme has been expanded to 62 separate spe-cies compared to the more common 25, or fewer,species included in most schemes (8, 25). Inpreliminary trials, these rapid methods haveproven to be readily adaptable to the clinicallaboratory routine. Almost all of the speciesidentifications are accomplished with the assim-ilation, fermentation, and urease tests afterdetermining that no sexual forms are present(asporogenous yeasts) and after recognition ofwhether or not hyphal structures are produced.There are only four instances requiring differen-tiation by growth at 20 or 37 C (Cryptococcusneoformans versus Cryptococcus luteolus; T.sphaerica versus T. candida; T. bovina versusT. pintolopesii; C. krusei versus C. slooffii),and these can be resolved without subcultur-ing. For example, with Cryptococcus neofor-mans and Cryptococcus luteolus, the fermenta-tion tests are incubated at 37 C and Cryptococ-cus neoformans produces acid (no gas) fromglucose, whereas Cryptococcus luteolus wouldshow no change in the color indicator since itdoes not grow at 37 C. Similarly, with C. kruseiand C. slooffii, the former would be positive forglucose assimilation at room temperaturewhereas the latter would be negative, althoughit would produce gas from glucose at 37 C. Arequirement for differentiation by additionalmorphological studies occurs in only five cases,and in at least two of these the additional stud-ies might not be necessary; i.e., C. humicola isalways urease positive whereas Trichosporoncutaneum is variable, and C. lambica alwaysproduces gas from glucose whereas Trichospo-ron penicillatum usually is negative.The YNB method possesses several advan-
tages over the Wickerham method in additionto yielding results more quickly. The colorchange occurred very early (usually after over-night incubation) and was an added aid in ob-taining rapid results. Adams and Cooper (1)reported that the color change in their methodwas "...synonymous with growth..."in everytest completed at that time. Furthermore,growth could be read quite easily by 48 h asseen in Fig. 4, where the printing on the under-lying template is sharp at disks where growthdid not occur but hazy at disks where growthwas present. Obviously, any carryover of asource of carbon with the inoculum is not aproblem with the YNB method, since very lit-tle, if any, growth occurred around disks con-taining a carbohydrate which could not be uti-lized by the species tested. Additional advan-
tages of the YNB method are economy andrapid preparation once disks have been pre-pared and stored. A single plate is used com-pared to 12 tubes for the Wickerham method,and these plates can be prepared in one-halfthetime required for inoculation of the 12 tubes inthe Wickerham method.At this point we do not know how strains
with latent assimilation reactions according toLodder (15) will behave when a very dense inoc-ulum is used. Allowance for this has been pro-vided in the present scheme for identification(Table 3) by including the species twice, oncefor a negative reaction and again for a positivereaction (e.g., Cryptococcus gastricus for lac-tose assimilation).
There are several points which should benoted with respect to the nitrate assimilationand fermentation tests. The nutrient used inthe nitrate test is acid, and during the first fewhours of incubation the area of basal mediumsurrounding the disks may change from a lightgreen color to pale yellow. Adding buffer to thebasal medium eliminated this but it also re-duced the sensitivity of the test, resulting insome false-negative reactions. This pale yellowcoloration is not apparent after overnight incu-bation and it has not influenced the results re-ported above. One should be aware, however,that this early change in color does not indicatethat growth has occurred. A similar early colorchange may occur around disks in the carbohy-drate assimilation test, but this too does notaffect readings after overnight incubation. Thefermentation tests require the careful overlayof vaspar on the cell suspension to avoid trap-ping air between the two. Since these tubes areincubated at 37 C after preparation at roomtemperature, trapped air will expand andmight lead to a false reading as a bubble of gas.If trapped air is noted after addition of thevaspar layer, the position can be marked on thetube, and the test should be read as positiveonly if the vaspar plug is displaced more thanwould be expected from expansion of thetrapped air. This has not been a serious prob-lem because the plug is displaced at least sev-eral centimeters in almost all cases ofgas forma-tion. The vaspar must be maintained com-pletely melted and should not solidify untilafter being layered on top of the cell suspen-sion. This will insure a full seal by the vaspar.We have maintained the bottle of vaspar in ahot water bath while preparing tests.The pseudogerm tube test is an excellent pro-
cedure for presumptive identification of an iso-late as either C. albicans or C. stellatoidea, butinterpretation of this test by inexperienced tech-
nologists requires some caution. Dolan (5) re-ported negative results in the original test for17 of 133 (13%) strains of C. albicans. Afteridentification of species by additional tests,these strains were found to form relatively fewpseudogerm tubes which were missed initially.Our experience has been similar. In addition tothis potential for false-negative results, onemust reckon with the possibility for obtainingfalse-positive readings. Ahearn (2) has com-mented that "inexperienced personnel may mis-take germinating arthrospores of G. candidumor Trichosporon species for germ tubes of C.albicans." He points out also that among spe-cies of yeasts which can produce pseudohyphae"... colonies may have formed hyphal elementson isolation agar. These may be mistaken forgerm tubes when observed in serum." Buckleyand van Uden (2) also noted that some pseudo-mycelium might be carried over in the initialinoculum, although they were able to distin-guish this from true pseudogerm tube forma-tion. We agree with these observations thatinexperienced personnel may obtain false-posi-tive results since we have seen even Cryptococ-cus gastricus produce elongated (hyphal-like)blastospores still attached to oval mother cells.In one of our laboratories C. tropicalis wasisolated repeatedly from the blood and urine ofa patient who had open heart surgery. Thisstrain of C. tropicalis produced elongated pseu-dohyphal cells in serum within 3 h and thesewere grossly similar to pseudogerm tubes. Onemust recognize the possibility that this mayoccur more frequently as the identification ofyeasts involved in opportunistic infections be-comes more precise. Unfortunately, there is evi-dence that some laboratories rely exclusivelyon this test for identification of C. albicans (14,20), and this cannot be justified. In our opinion,a positive pseudogerm tube test can be consid-ered presumptive identification of C. albicansor C. stellatoidea to be confirmed by additionaltests, whereas a negative test does not excludethe identification of these two yeasts.The sequence to be followed for identification
of yeasts is readily adaptable for use in clinicalmicrobiology (Fig. 9). Since prepared disks andbasal media can be stored, the tests can be setup rapidly. In our experience, disks stored in adesiccator have been satisfactory for up to 6months, with the exception of xylose. When thexylose disk turns brown, it is no longer reliable.There is some advantage to performing the testfor hyphae or pseudohyphae directly from sev-eral isolated colonies on the primary isolationmedia to determine whether more than onespecies is present. One more day would be re-quired, but additional information would be
IPRODUCTIOOF HYPHAEPSEUDOHY
(1-2 days)
SPECIMEN
LABORATORY
ISOLiTEDYEAST COLONIES
MEDIUM FOR HARVESTING(1-2 days)
)N ~~~~~PSN~~~-'OR(PHAE HEAVY
SUSPENSION
RAPID TESTS (1-2 Days)
I3EUDOGERMTUBE TEST
(3 h)
1. CARBOHYDRATE ASSIMILATION2. NITRATE ASSIMILATION3. FERMENTATION4. UREASE PRODUCTION
FIG. 9. Sequence of procedures for identificationof yeasts.
obtained in some cases. The inoculum for theharvesting medium could be obtained from thesurface of the CMT agar plate since it has notbeen covered with a glass cover slip.Our schema is the same in principle as that
reported by Barnett and Pankhurst (3), i.e.,identification of asporogenous yeasts based pri-marily on physiological tests and multiple en-tries of a species when results of a test arevariable. These authors also used the data pub-lished by Lodder (15) for 341 species and addeddescriptions of 93 new species for a total of 434.Their key to the identification of these yeastswas constructed after computer analysis re-vealed the most efficient tests for differentia-tion among species. This publication is recom-mended for those laboratories interested in anexpanded schema for identification of aspo-rogenous yeasts.We recognize that a search for, and identifica-
tion of, yeasts with all specimens is not practi-cal for clinical laboratories with workloads al-ready strained. Communication and coopera-tion between physician and microbiologist isrequired to identify cases in which recovery ofyeasts would bear significantly on clinical man-agement of the patient. The need for this collab-oration has been expressed clearly by Klainerand Beisel (12): "In this situation, a close recip-rocal relationship must develop between theclinician at the bedside and the microbiologistin the laboratory. The physician must alert themicrobiologist to the possibility of an opportun-istic infection; in return, the microbiologistmust regard isolation by culture of 'contami-nants' or saprophytes, especially if recurring,with caution and suspicion. Together, such a
medical team must attempt to determine thesignificance of the presence of these orga-nisms."
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