Rapid methods for identification of yeasts

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JUlY p.

Rapid Methods

for

Identification of Yeasts

M. HUPPERT,* GLORIA HARPER,' SUNG H. SUN, AND V. DELANEROLLE Mycology Research Department, Veterans Administration Hospital, Long Beach, California 90801

Received for publication 10April 1975

Opportunistic infections by yeasts have been implicated as one of the major causes of complications in the compromised patient. Rapid recognition and identification of these yeasts is essential for patient management, but conven-tional liquid medium methods for completing identification tests are cumber-someandtimeconsuming. Rapid tests have been devised based on modifications

of methods commonly usedinbacteriology. Theserapid methods included tests

forcarbohydrate andnitrate assimilation, fermentation,andurease production.

These werecompared with several current methods for accuracy of results, for

time to finalidentification, andfor economy of time and reagents. In addition, the usual tests for pseudogerm tube formation, for production of hyphae or

pseudohyphae, and for growth temperatures were included. The rapid tests

achieved96% orbetteraccuracy compared with expected results, and 46 species

ofyeasts were identified in 1 to 2 days compared with the 10 to 14 days required

by conventional liquid culture methods.

Modern medicine, with the capacityfor

pro-longing thelife of a patient, has seen a marked

increase in secondary infections among pa-tientswhoseresistance has beeencompromised

by debilitating chronic disease, by therapeutic

modalities, andby severe injury. Fungi are a

common causeofinfectionsinsuchpatients (7, 9, 11, 12, 14, 16, 19, 24). This increased fre-quency inthemycoseshas beennotable in two

additionalrespects: greater severityofthe

dis-easeandgreaterdiversityinetiologicalagents (2, 8, 10, 16, 21, 22, 25). Yeasts are among the most commonetiological agents.

Since many speciesoftheseyeastshad been

consideredinnocuouspreviously, theirrecovery

from a pathological specimen presents

prob-lems forthe laboratory which must identify a

variety ofisolates and for clinicians whomust

judge their significance. Conventional liquid

culture methods for speciation ofyeasts are

cumbersome and time consuming (15, 25, 27,

28). Rapid methods havebeenproposedbut

re-strictions in the number of tests used limits

identificationto relatively few species (1, 5, 6,

8). In addition, mostclinical laboratories lack

personnel with more than minimal training

andexperience inidentifyingfungi.

The presentstudyhas beenlimitedto identifi-cationof yeastsinthegeneraCandida, Crypto-coccus, Torulopsis, and Trichosporon.

Geotri-chum candidum, although not a yeast, has

been includedbecause itoccursfrequentlywith

'Present address: Clinical Laboratory, Little Company ofMaryHospital,Torrance,Calif.90503.

yeasts but canbe differentiatedfrom thelatter

by the same procedures. Two objectives were

planned. The first involved the development

and evaluation ofrapid methodsforidentifying

almostallspecies of yeasts recovered from

hu-mans(15). The second required thatthe

meth-ods should be familiar to technologists

compe-tentwithonlybacteriological techniques. Since many ofthese personnel lack experience in

my-cology, thetestsemployed are almost all

physio-logical, and morphological criteria have been

limitedprimarilytoobservation of whether

hy-phae (orpseudohyphae) arepresent or absent.

Amostessential firststep for speciating yeasts

is recognition of whether or not perfect stage

forms are present, e.g., asci and ascospores.

Descriptions of these are beyond the scope of thepresentstudybut areavailableinreference texts (8, 13, 15, 25), or such cultures can be

referred to a zymologist for identification.

Whennoinformationabout sexual sporeforms

is available, all yeast identifications must be

considered presumptive.

(This paper includes,inpart, a thesis

submit-tedbyG. H.totheCalifornia State University, Long Beach, in fulfillment of the requirements for the Master of Sciencedegree.)

MATERIALS AND METHODS

Theprocedures describedinthis section represent theoptimal methodsderivedfrom thisstudy. Varia-tionswhichwere studiedarepresentedinlater

sec-tions.

Strains used. Forty-sixpreviously identified spe-21

on February 6, 2020 by guest

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22 HUPPERT ET AL.

cies (Table 1) were maintained as stock cultureson

yeast-malt agar (8, 15).

Preparation of inoculum. Dense suspensions wereprepared from confluent growth at room

tem-perature (24to 48 h) using6ml of sterile waterper petri dish (100 by 15 mm) followed by transferring the suspension to a sterile tube (16-mm diameter). Although quantitative standardization was per-formed by optical density (OD) determination and

TABLE 1. Listof46speciesstudied

Species Sourcea

Candida albicans ... ATCC 18804

C. ciferrii ... CBS4856

C. curvata

...

ATCC 10567

C.guilliermondii ... ATCC6260

C. humicola ... ATCC 14438

C.intermedia ... ATCC 14439 C. kruseii ... ATCC6258

C. lambica ... ATCC 9330

C. lipolytica ... ATCC 18942

C. lusitaniae ... CBS 4413

C. membranaefaciens .... CBS 1952

C.norvegensis ... CBS 1922

C.parapsilosis ... ATCC22019

C.pelliculosa ... Pablo Negroni C.pseudotropicalis ... ATCC4135

C. ravautii ... ATCC 18821

C.rugosa ... ATCC 10571

C. silvae ... CBS 5498

C. stellatoidea ... ATCC 11006

C.tenuis ... ATCC 14462

C.tropicalis ... ATCC 14056

C. utilis ... ATCC 22023

C.zeylanoides ... ATCC 10674 Cryptococcusalbidus .... ATCC 10666 C.gastricus ... CBS 1927

C. Iaurentii ... ATCC 18803

C. neoformans ... OrdaPlunkettM443

C. terreus ... ATCC 11799

C. uniguttulatus ... CBS1730

Torulopsisbovina ... CBS 2760 T. candida ... ATCC 12790 T.etchellsii ... ATCC 11504 T.glabrata ... ATCC 15545 T.holmii ... ATCC 22034 T.inconspicua ... ATCC 16783 T.magnoliae ... ATCC 13782 T.norvegica ... CBS 4239 T.sphaerica ... ATCC 2504 T.stellata ... ATCC 10673 T. versatilis ... ATCC 20222

Trichosporoncapitatum . ATCC 10663 T. cutaneum ... ATCC 13445 T.inkin ... ATCC 18020 T.penicillatum ... ATCC 18019

T.pullulans ... ATCC 10677

T. variabilis ... BNCYC Endomycopsis

chodati

by cellcount, apractical and satisfactoryestimateof cell density wasobtained whenthesuspension com-pletelyobscured the blacklines on astandard Wick-erham card (3 India ink lines approximately

0.75-mm wide on awhitecard). The less dense inoculum required forsome ofthe conventional methods

em-ployed for comparison with the rapid methods was

achieved by diluting the dense inoculum until the black lineswerevisibleasdarkbands.

Carbohydrate assimilation. Thefollowing carbo-hydrateswereused: glucose, maltose, sucrose, inosi-tol, lactose, cellobiose, raffinose, melibiose, erythri-tol, xylose, galactitol (dulcitol), and trehalose. Thesewereselected onthe basis of critical value for differentiating among species (15). The purestgrade reagents availablewereused.For ourmodified

aux-anographic method, dry disks containing carbohy-drateplusnutrient wereprepared. Others have used disks saturated withcarbohydrate only (1, 5, 25). A lOx concentration (8.04 g/120 ml ofdistilled 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 adding2g ofcarbohydrateto 10ml ofthe

lOx concentrate of YNB and dissolving with mild heating. Solutions were sterilized by passage through a membrane filter (0.20 ,um) followed by aseptic transfer to sterile vaccine vials. A layer of sterile blank 0.25-inch (ca.0.64-cm)disks (concentra-tiondisks, Difco)inpetridishes wassaturated with

oneof the carbohydrate solutions by dropwise

addi-tionfromasyringe and needle. These weredried48

to72h in an evacuated desiccatorcontaining anhy-drous CaSO4. The disks containing inositol, raffi-nose,andmaltoserequiredasecondsaturationafter thefirstdryingperiod.

The basal agar for the carbohydrate assimilation

test contained agar (20 g/1,000 ml), bromothymol blue (0.16 g/1,000 ml), and phosphate buffer (100 ml/1,000 ml). The phosphate buffer stock solution

was a 1:1 mixtureof0.067 MNa2HPO4 and 0.067 M KH2PO4 (pH 6.8). The melted agar was dispensed while hotinto 2-oz. (ca. 0.06-liter)screw-cap bottles (approximately30ml/bottle). Volumes of 30to50 ml

can be used, but the smaller volume enhanced the final reading of growth becauseofthe thinner layer ofagar. The bottles ofagar were autoclaved for 15 min at 121C. Iftightly capped after cooling, these bottles can be stored indefinitely at room

tempera-ture.

The assimilation test was performed by pouring

onebottle ofmelted agar for each strain to be tested

into sterile petri dishes (150 by 15 mm). After hardening, the plates were streaked for confluent growth with a swabsaturated in the dense

suspen-sionofcells.Thestreaked plates were allowed to dry before applying the disks, usually 30 min. Disks

were applied to the surface of the agar using a template (Fig. 1) under the dish to insure placing

thedisks equidistant apart. Theposition of the

glu-cose disk was marked on the bottom of the petri

dish. Plates were incubated at 25 C (room tempera-ture) and read daily for 3 days. The earliest

indica-tion of apositivereactionwas achange of the indica-'ATCC,American Type Culture Collection; CBS,

Centralbureau voor Schimmelcultures; BNCYC,

British National Collection of YeastCultures.

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tor color from blue-green to yellow surrounding the disk. This could be confirmed later during the incu-bation period by enhanced growth of the organism in the area surrounding the disk. Confirmation by reading forgrowth may be required with those orga-nisms whichproduce acid from many carbohydrates, turning large sections of the plate completely yel-low. Under these circumstances, if no enhanced growth is evident around individual disks, the test

is negative for that carbohydrate. This procedure willbe referred to as the YNB method in subsequent

sections.

The results obtained by the YNB method were compared withthose obtained from three additional methods. The Wickerham liquid medium method was performed without agitation according to the procedure described in the Lodder text (15). The oxidation-fermentation (OF) tube method followed theprocedures described by Webb et al. (27), except thatcommercially available OF media (Difco) was used. Tubesinboth methods received 0.1 ml of di-lutedinoculum. The last method used OF media in a petri dish. These were streaked heavily as in the YNBmethod, anddry disks containing only carbohy-drates were placed on the surface. Concentrations of carbohydrates were identical to those used in the YNBmethod. For the comparative studies, alltests were incubated at room temperature and read at intervals overa 14-day period.

Nitrate assimilation. Dry disks containing a

sourceofnitrogen plusnutrient werepreparedina

mannersimilartothat used forcarbohydrate assimi-lation.A 1%KNO3solution ina 4xconcentration of yeast carbon base (Difco) (15, 25, 28) wasused to

impregnate the disks. Disks for use as a positive control were saturated with a solution of 1%

(NH4)2SO4 in a 4x concentration of yeast carbon base. Disks for negative controlsorpersisting

endog-enousnitrogensourcecontainedonly4x concentra-tionof yeast carbon base. The methods for steriliz-ing solutions and for saturatsteriliz-ing anddrying the disks

werethesame asdescribed forcarbohydrate

assimi-lation. The basal agar fornitrateassimilation

con-sisted of agar (20 g/1,000ml) with bromothymol blue (0.08g/1,000 ml).Nophosphatebufferwasaddedto thismedium, andadjustmentto alight green color (approximately pH 6.8) may be required. The

me-dium wasdispensed in approximately 20-ml/bottle

amountsand autoclaved for15minat121C. Testing for assimilation ofnitrate was required for everyspeciesaccordingtothe schemedeveloped. One bottle for each strain wasmelted and poured intosterile petri dishes (100by15mm). Plateswere streakedwithdense inoculumanddried,and appro-priatediskswereaddedinthemannerdescribed for carbohydrate assimilation. Incubation was at 25C

for24 to48 h. A positive nitrate assimilation test

was indicatedbyacolorchangefromlightgreento dark blue surroundingthe disk containing KNO:1. The color change was caused by the residual, strongly alkalineK+afterreduction of theNO:r-.A

colorchangetobrightyellowdevelopedaroundthe (NH4)2SO4 disk, resulting from utilization ofthe NH4, and anacidreactionfromresidualSO4=

com-FIG. 1. Template placed underpetridishfor spot-ting disks and reading results. Abbreviations for

carbohydrates (clockwise forouterand innercircles) correspond to sequence in Table3 beginning with glucose.

bined with acidfromoxidation of the glucose in the yeastcarbon base.

The conventional methods used for comparison

were the Delft auxanographic plate (27) and the Wickerham liquid medium procedures (8, 27). Di-luted inoculum (0.1 ml) was used, and incubation for bothwas atroomtemperature. The Delft plate test

wasreadat 48and96h,and the Wickerham test was readdaily for7days. A positive by the latter method required subculturing to a second tube to determine whether growth in the first tube could have been causedby carryover ofa sourceof nitrogen.

Fermentation tests. One milliliter of the heavy suspensionwasplacedineachof three1

1-mm-diame-ter testtubes. (The tubes need notbe sterile.) Glu-cose, maltose, and sucrose fermentation tablets (Key ScientificProducts, Los Angeles, Calif.) were

addedone to atube. One milliliter of molten vaspar

was added to each tube carefully with a Pasteur pipetteso as not totrapanyairbubbles. The vaspar

was a mixture (1:1, wt/wt) ofpetrolatum jelly and paraffin (melting point,48 to 56C). The tubeswere

incubated at37C for24h. Apositivefermentation

reaction wasindicatedbyariseofthe vasparplug, whichseparatedcompletely from the liquid

suspen-sionbecause of gasproduction.

Results with this rapid fermentation tests were

compared with those obtainedby the conventional

Durham invertedtubeprocedure (8, 27). Tubes re-ceived 0.1 ml of diluted inoculum and were

incu-batedat roomtemperaturewithfinalreadingsat 14 days.

Urease production. One milliliter of the heavy suspension was placed in an 11-mm-diameter test tube (nonsterile). One urease tablet (Key Scientific Products)was added, andthe tubewasincubatedat

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37C for 24h. Abright pink color at the endofthe incubation perioddenoted a positive reaction.

The conventional method used for comparison was Christiansen urease agar slants (8, 27). Slants

wereinoculated with 0.1 ml of the dense suspension, incubated at room temperature, and read daily for 5 days.

Pseudogerm tube test. Most investigators refer to thisasthegerm tubetest. Mackenzie(17, 18)has stated that the termgermtube". isamisleading

one, asthe similarity to the truegerminative 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 of

the surface of the parentcell and absence of

proxi-mal constrictions. ." (17). All strains were studied for pseudogerm tube formation by adding 1 drop of

thedense inoculum to 0.5 ml of fetalbovineserumin

a testtube (10 by 75mm) (8). Thetubes were incu-bated at 37 C and read at 3h for the formation of

pseudogerm tubes.

Production of hyphae or pseudohyphae. The presence orabsence ofhyphae orpseudohyphae was

determinedin corn mealagar(withoutglucose) con-taining 1% Tween 80(CMT)inapetri dish (50by12

mm). The inoculumwas obtained fromthe harvest-ingplate prior topreparingthe dense suspension. A

smallamountofgrowthon astiff needle waspressed through theagar to the bottom of theplateand then

drawn throughthe agarat anangletomakealong

cut.Incubation was atroomtemperature for 24to 48

h. Production ofhyphae wasobserved by inverting

theplate on the stage of the microscope and viewing withthe low-power(10x)objectivelens.

Additional tests.For some species, temperature

tolerance(i.e., growthat 20and37C)wasrequired for identification. The 20C tolerancewas observed

usingthe yeast-maltagarplateprior toharvesting.

An additional plate ofyeast-malt agar was

incu-batedat 37Cwhen this informationwasneeded for

final identification. Ifmorphology was required to

differentiate among several species, microscopic examination was made on the surface of the CMT

plate.

RESULTS

Density of suspension. Since rapid results with these new procedures required a heavy inoculum, it was necessary to determine the upper and lower limits of inoculumdensity for obtaining accurate and reproducible results. The wavelength (Coleman junior spectropho-tometer, model 6D) yielding maximum sensitiv-ity fordetecting a difference in density of cells was determined using two dilutions of selected species from the genera Candida, Cryptococ-cus, Torulopsis, and Trichosporon. The ODs for these dilutions were read at several wave-lengths in a scan of the visible spectrum. The wavelength yielding the greatest difference be-tween the two curves was chosen as the one

providingmaximum sensitivity for these yeast

cell suspensions. A typical exampleappears in Fig. 2. The wavelengthsof maximum sensitiv-ity for all species clustered around 580 nm. Upper limit density studieswereperformed by harvesting successive platesofthe several spe-cies with the initial 6 ml of water until the suspension attained a viscosity precluding the harvesting of additional plates. This and sev-eral serial dilutions were used to perform the carbohycirate and nitrate assimilation, urease production, and fermentation tests. Accurate, reproducible, and rapid results were obtained with all suspensionsyieldingan OD - 0.6

(ap-proximately 0l cells/ml), andit wasconcluded that only a minimum limit for inoculum den-sity was required. A suspension in a 16-mm-diameter tube which obscures the lines on a Wickerham card has an OD - 0.6, and this

simplecard method was used routinely. Carbohydrate assimilation. The four meth-ods were evaluated in terms ofpercentage of agreement withexpectedreactions for each spe-cies as published in Lodder (15), referred to as the "reference."The combinedresults for all 46 species with each of the four methods are

pre-01

0

8-0 0 6-F-1

06 LL

-j

0

0Q4-0L

0

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 for detecting differences in density of cells in a

suspen-sion.Specieswas C.guilliermondii.

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RAPID

sented in Table 2 and Fig. 3. The rapid YNB method produced 90% of the expected results within 24 h and 97% agreement with the refer-ence by 48 h. The Wickerham method required at least 3 to 5 days to attain the same level of agreement as the YNB procedure, although the former achieved 100% agreement by 14 days. This was expected since the results published in the reference were obtained with the Wicker-ham method. The two procedures using OF media were not as good. The OF plate method was particularly unsatisfactory because color

changes began to revert onday 3 fromyellow

back to blue as the carbohydrate was ex-hausted, peptone was utilized as a carbon

source, 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 four methods for assimilation of carbohydrates

Incubation time(days) Method

1 2 3-5 6-14

OF,plate 82 ± 13" 86 ± 15 86 ± 15 86 ± 15

OF, tube 83 11 89 9 93 5 93 5

YNB 90 ±7 97 2 98 t2 98 2

Wicker- 78 ± 11 88± 10 98 ± 3 100± 1

ham

___ _ _ _ _ I~~~~~~~~~~~~~~~~~~~~~~~~~~~

"Results are presented as percentage of agree-mentwith reference (15) -+- standard deviation.

COMPOSITE COMPARISON OF CARBOHYDRATE

l00o, ASSIMILATION TESTS

90_ 1

80_

70-60

3-5

TYIME-DAYS

FIG. 3. Comparison of composite results obtained by four methods for assimilation of carbohydrates expressed aspercentage of agreement, withexpected

results as published in reference 15. Symbols for bars: dotted, OF plate method; cross-hatched, OF

tube method; striped, YNB rapid method; solid, Wickerhammethod.

Fig.3donotreflectthis reversion for the 3- to

5-day and 6- to 14-day periods, but only that a

maximum of 86% agreement had been obtaina-ble by this method. In preliminary studies YNB

had been usedin the mediuminplates rather

than in the disks which contained only the

carbohydrates. The same reversion of

indi-catorcolor occurred,and this was discarded in

favor of incorporating nutrient and

carbohy-drateindisks. Inthe latter method, reversion

of indicator color did not occur (Fig. 4). The YNB method was less variable during

the first 2days than the other methods (Table

2). The principal cause for this variability is

illustrated in Fig. 5-7 for the three

carbohy-drates trehalose, cellobiose, and raffinose. The percentage of agreement with the reference

after24hisrelatively low for all three of these

carbohydrates by each of the four methods. By

48h, however, the YNB method achieved 96%

orbetteragreement with these three

carbohy-drates, whereas the other methods attained only 82% at best by this time. These results indicate that factors which presented problems

forthe conventional liquid media methods had

relatively little effect on the rapid YNB method

forcarbohydrate assimilation.

Fermentation tests. The rapid method and conventional Durham tube fermentation tests were evaluated in terms of the published ex-pected results (15) for each species. The rapid test (Fig. 8) produced accurate results in 1 day with 94% or better of the expected reactions, whereas the conventional Durham tube test

required 14 days for comparable agreement.

Two-thirds of the 6% incidence of

nonagree-mentusing glucoseinthe rapid test werewith

specieswhich yielded positive reactions when a

negative test was expected. This may have

re-sulted from incubation at 37 C with the rapid

procedure, since the reactions reported in the

reference were based on incubation at 25 to28C

for 24 days. In addition to yielding results

quickly, the rapidtest was very easy to read.

When fermentation had occurred, the vaspar

plug had been raised several centimeters in

almost everyinstance.

Nitrate assimilation. A comparison of

re-sults obtained by the rapid method andby the

Delftand Wickerhamprocedures indicated100%

agreementwith the referencebyall three

meth-ods. Incubation timefor the YNBmethod was

significantly shorter, 1 to 2daysversus4 to 14

days. The change in indicator color fromlight

green todark blue around theKNO3 diskwas obvious anddramaticwithallspecies

assimilat-ing nitrate, but either no change or a slight

yellowing occurredwithspecies whichwere

neg-ative for nitrateassimilation.Thedisk

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26 HUPPERT ET AL.

FIG. 4.

at 18h).

Typicalexample of carbohydrate assimilation resultsby the YNB rapid method (C.guilliermondii

Pseudogerm tube production.Asexpected, C. albicans and C. stellatoidea produced

pseu-dogermtubes consistentlywithin the 3-h

incu-bationperiod. However,C.tropicalis,C.

parap-silosis, and Cryptococcus gastricus also

pro-ducedstructures whichresembledpseudogerm

tubes. Although these were elongated blasto-spores, personnel inexperienced with the mor-phology of yeastsmightnot recognizethe 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 bar

graphs.)

ing(NH4),S04wassurroundedbyayellowzone

with all species, and the area around disks

containingyeast carbon base without asource of nitrogen either remained light green or

showed a slight change to yellow. Therefore,

carryoverofendogenousnitrogendidnot

influ-enceresults withthe rapidtest, and therewas

no need for starving cells or subculturing. In

fact,themedium controldiskwasnotrequired.

< 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 bar graph.)

90

8o4_

TREHALOSE

mI

0

I

i

£

V 70.

606

50.

CELLOBIOSE

I

100|

'10

t.

I E

6 14 1

I I

All.

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RAPID IDENTIFICATION OF YEASTS 27

sult. In our opinion complete relianc

pseudogerm tube test for identifying

cans orC. stellatoideaby inexperience nel maynot be justified (seeDiscussil Hyphae or pseudohyphae forma species in the generaCryptococcus and sis failed to form any structures re

hyphalformation in the CMT agar. In

allCandida and Trichosporon specieE

strain ofG. candidum were positive. I

nize that occasional strains of C.

mondii and C. parapsilosis may not

100_.

90+_

80_t

RAFFINOSE

I

S

"I

i

2 3

TIME-DAYS

FIG. 7. Results obtained by four metho similation of raffinose. (See Fig. 3 for k

graphs.)

ce on the obvioushyphal structures, and this has been

C. albi- considered in the construction of our schema

d person- foridentification of theseyeasts(seebelow).

On). Urease production. A comparison of results Ltion. All obtained by the rapid method and by the con-Torulop- ventional method showed 100% agreement with

sembling the reference by both methods. The principal

contrast, advantage of the rapid method was the much s and the shorter incubation time, i.e., 1 day versus 5 We recog- days.

guillier- Quality controls for assimilation, urease,

produce and fermentation tests. Satisfactory quality

control for these tests required a suspension yielding positive reactions and one resulting in negative tests. Nosinglespeciesproduced

posi-tiveresultsinalltests.Severalstrains in

combi-nation were tried, and the best results were obtained with C. pelliculosa andCryptococcus

laurentii. Each species was grown and

har-vested separately and then testedfor

satisfac-tory

inoculum

density. Equal aliquots

of each

suspension were mixed, and the mixture was

used to perform the rapid procedures. All the

rapidtests weredefinitelypositivewithin48h.

Ifpreferred, suspensions of these two species

could be usedseparately rather than mixedinto

asinglesuspension. OurstrainofCryptococcus

laurentii assimilated all 12 carbohydrates and

was urease positive. The C. pelliculosa strain produced positive reactions in tests for nitrate assimilation and fermentation. A suspension of Eds for as-

Trichosporon

capitatum

wasusedas a

negative

iey to bar control. Our strain of this

species

assimilated

only glucose andwasnegative inall othertests.

GLUCOSE

100I

90~~~~~~~~I

3..80-LU

MALTOSE SUCROSE

TIME - DAYS

FIG. 8. Comparison ofresults obtained by therapidand bytheDurham tubefermentation testsexpressed

aspercentage ofagreement, with expected results aspublished in reference15. Symbols: striped bar, rapid method; solidbar,Durham tubemethod.

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28 HUPPERT ET AL.

Schema for identification of yeasts. This series oftests would not be complete without presenting a schema bywhich theresults could beused easily for identification ofthese imper-fectyeasts. A schema has been constructed for thispurpose(Table3). Itincludes allspecies in the genera Candida, Cryptococcus, Rhodoto-rula, Torulopsis, and Trichosporon which,

ac-cording to Lodder (15), have been recovered

from humans or animals. Inaddition,

Saccharo-myces cerevisiaeand G. candidum (notayeast)

have been includedbecauseof the needto

distin-guish these commonfungi from species in the

above five genera.

The principal objectiveguiding the

construc-tion ofthis schema was to include only those reactions which were most important for differ-entiating amongspecies, keepinginmind that

many personnel in clinical laboratories have

little experience with these organisms. A

pri-mary separation was made on the basis of

whether hyphae or pseudohyphae were either

well developed or absent (or rudimentary) as

seen in the CMTagar. Thetwospecies of

Can-dida (C. guilliermondii and C. parapsilosis),

inwhichoccasionalstrainsfailtoproduce

pseu-dohyphae, appear under bothcategories.

Simi-larly, occasional strains ofR. glutinis andR.

rubra may produce pseudohyphae, and these

appear in both sections of the schema. Species

with variable reactions in some tests appear

several times, when the result with a single

reagent might be positive and again when it

would be negative. Therefore, decisions in

al-mostallcaseswerereducedtosimply positive

or negative results. Groups of species were

formed on the basis ofassimilation tests for

nitrate, glucose, maltose, and sucrose. These

groups could be differentiatedinto species

by

reading results foronly those additional tests

whichwerecriticalwithin the group.

Prelimi-nary trials with this schema bytechnologists

unfamiliar with the yeasts have met with

general acceptance and have resulted in

suc-cessful species identifications ofyeasts

previ-ously reportedincompletely,e.g., unidentified

yeasts or Candida species that are not C.

albicans. An additional benefit is gained by

includingonlycriticalreactions inthe schema.

Almost all the reactions responsible for the lack of agreement between the rapidmethods

andthe reference have beeneliminated.

DISCUSSION

Achievements by modern medical science

have produced a population of people uniquely

susceptibletoinfectionbymicroorganisms

pre-viously considered innocuous. Some of these

organismsexistsaprophytically inman's

envi-ronment,whereas others occurcomensally,and the individual withcompromiseddefenses can-not escape eventual exposure. Theyeasts, as a group, 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 stated

that "only 30% of disseminated candida

infec-tions are diagnosed correctly antemortem so

thatimprovedmethods ofdiagnosisareclearly

needed." There is ample evidence, however,

thatnotonly morbidity but also mortalitymust

besignificant. Law, MacMillan,and their

asso-ciates (14, 19) reportedstudies on 427burn pa-tients. Candida species were recovered one or more timesfrom63.5% ofthepatients. In addi-tion to positive cultures from thewound, Can-dida species were found in urine (48%) and in

blood (5%),and, of the65patients whodied, 14

(22%)deathswere attributabletodisseminated

candidiasis. Miller et al. (20) reported that in

the Center for the Study of Trauma at the

University ofMaryland 16% ofthe nosocomial

infections werecausedby Candida species, ex-ceeded only byPseudomonas infections(24%).

Thesewereprimarily shocktraumaand

postop-erativethoracicsurgerypatients, withonlyan

insignificant numberofburncases.Gainesand

Remington(7)reviewedsurgical records for the

1960-1967period and found42casesdeveloping

systemic candidiasis, with 19 deaths. They

pointedoutthatcandidiasiswas notdiagnosed

early enoughforappropriatetreatment inmore than50%. Rifkind etal. (24)described107cases receivingrenal transplants, with51deaths. Sys-temicmycoses wereinvolvedin 23 (45%),

Can-didaspeciesaccountingfor 14. Hutter and

Col-lins (9) found 202 mycoses in cancer patients,

withyeasts responsible for 130. Eighty-four of

the latter occurred inthe leukemia-lymphoma

group. Kahanpaa (10), in anextensive survey

involving8,290 specimens, identified a total of

35 species of yeasts in 21% ofbronchial

secre-tions, 34% of lung tissue specimens, 71% of

autopsylungspecimens, and 20% ofthe pleural effusions. One mustconclude that yeasts repre-sent asignificant problem in these diseases and thatearly recognition is essential to insure ap-propriate therapy.

Ourapproach to the developmentof methods

foridentifying yeasts has recognized these

re-quirements. The procedures are adapted almost

entirelyfrom those familiar to microbiologists

withlittleexperience in mycology, and the re-sults areobtainedrapidly, usually in2 to 4 days J. MICROBIOL.

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after primary isolation compared to 10 to 14 days by conventional liquid culture methods. In addition, the presumptive identification scheme has been expanded to 62 separate

spe-cies comparedto the more common 25, orfewer,

species included in most schemes (8, 25). In

preliminary trials, these rapid methods have proven to be readily adaptable to the clinical laboratory routine. Almost all of the species identifications are accomplished with the

assim-ilation, fermentation, and urease tests after

determining that no sexual forms are present (asporogenous yeasts) and after recognition of

whetherornothyphal structures are produced.

Thereareonly fourinstancesrequiring

differen-tiation by growth at 20 or 37C (Cryptococcus

neoformans versus Cryptococcus luteolus; T.

sphaericaversusT.candida;T.bovinaversus

T. pintolopesii; C. krusei versus C.

slooffii),

and these can be resolved without

subcultur-ing. For example, with Cryptococcus

neofor-mansandCryptococcus luteolus, the

fermenta-tiontests areincubated at37Cand Cryptococ-cus neoformans produces acid (no gas) from

glucose, whereas Cryptococcus luteolus would

show no change in the color indicator since it

doesnotgrowat37C.Similarly,with C. krusei

and C.

slooffii,

the former would be positivefor

glucose assimilation at room temperature

whereas the latter would be negative,although

it would produce gas from glucose at 37C. A

requirement for differentiation by additional

morphological studiesoccurs inonly five cases,

andin atleasttwoof these the additional

stud-iesmightnotbe necessary; i.e., C. humicolais

always urease positive whereas Trichosporon

cutaneum is variable, and C. lambica always

produces gas fromglucose whereas

Trichospo-ronpenicillatum usually is negative.

The YNB method possesses several

advan-tagesover the Wickerham method inaddition

to yielding results more quickly. The color

change occurredveryearly(usuallyafter

over-night incubation) andwasanadded aidin

ob-taining rapid results. Adams and Cooper (1)

reportedthat thecolorchangeintheir method

was"...synonymous withgrowth..."in every

test completed at that time. Furthermore,

growth could be read quite easily by 48h as

seeninFig.4, where theprintingonthe

under-lyingtemplate issharp atdisks wheregrowth

did not occur but hazy at disks where

growth

was present. Obviously, any carryover of a

source of carbon with the inoculum is not a

problem withthe YNBmethod, since very

lit-tle, ifany, growth occurred around disks con-taining acarbohydratewhichcould notbe uti-lized by the species tested. Additional

advan-tages of the YNB method are economy and

rapid preparation once disks have been

pre-paredand stored. A single plate is used

com-paredto 12 tubes for the Wickerham method,

and these plates can be prepared in one-half the

timerequired for inoculation of the 12 tubes in

the Wickerham method.

At this point we do not know how strains

with latentassimilation reactions according to

Lodder(15)willbehave whena verydense

inoc-ulumisused. Allowance for this has been

pro-vided in the present scheme for identification

(Table 3) by including the species twice, once

foranegative reactionand again forapositive

reaction (e.g., Cryptococcus gastricus for

lac-tose assimilation).

There are several points which should be noted with respect to the nitrate assimilation

and fermentation tests. The nutrient used in

the nitrate test isacid, and during the first few

hours of incubation the area of basal medium

surrounding the disksmaychangefrom a light

greencolortopale yellow. Adding buffer to the

basal medium eliminated this but it also

re-duced the sensitivity of the test, resulting in

somefalse-negative reactions. Thispaleyellow

colorationisnotapparentafterovernight

incu-bation andithasnotinfluenced the results

re-ported above. One should be aware, however,

that this early changeincolor does not indicate

that growth has occurred. A similar early color

changemay occuraround disksin the

carbohy-drate assimilation test, but this too does not

affectreadings after overnight incubation. The

fermentation tests require the careful overlay

of vaspar onthe cell suspensionto avoid

trap-pingairbetween the two. Since these tubes are

incubated at 37C after preparation at room

temperature, trapped air will expand and

might lead toafalse reading as a bubble of gas.

If trapped air is noted after addition of the

vaspar layer,the position can be marked on the

tube, and the test should be read as positive

only if the vasparplugis displaced more than

would be expected from expansion of the

trapped air. This hasnot been a serious

prob-lem because the plugisdisplaced at least

sev-eral centimetersinalmost allcasesofgas

forma-tion. The vaspar must be maintained

com-pletely melted and should not solidify until

after being layered on top of the cell suspen-sion. This will insure afull sealbythevaspar.

Wehave maintained the bottle ofvaspar in a

hotwater bath while preparingtests.

Thepseudogermtube test is anexcellent

pro-cedure forpresumptive identification ofan iso-late aseither C. albicans or C.stellatoidea,but

interpretationof this testbyinexperienced

http://jcm.asm.org/

(10)

30 HUPPERT ET AL.

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VOL. 2, 1975 RAPID IDENTIFICATION OF YEASTS 31

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32 HUPPERT ET AL.

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nologists requires some caution. Dolan (5)

re-ported negative results in the original test for

17 of 133 (13%) strains of C. albicans. After

identification of species by additional tests,

these strains were found to form relatively few

pseudogermtubes which weremissed initially.

Our experiencehasbeen similar. In addition to

this potential for false-negative results, one

must reckon with the possibility forobtaining

false-positive readings. Ahearn (2) has

com-mented that"inexperienced personnelmay

mis-take germinating arthrospores of G. candidum or Trichosporon species for germ tubes of C.

albicans." He points out also thatamong

spe-ciesof yeasts which can producepseudohyphae

"...colonies may haveformed hyphal elements

on isolation agar. These may be mistaken for

germ tubeswhenobservedinserum." Buckley

andvanUden(2) also noted thatsome

pseudo-mycelium might be carried overin the initial

inoculum, although they were able to

distin-guish this from true pseudogerm tube

forma-tion. We agree with these observations that

inexperienced personnel may obtain

false-posi-tiveresultssincewehave seen even

Cryptococ-cus gastricus produce elongated (hyphal-like)

blastosporesstillattachedto ovalmother cells.

In one of our laboratories C. tropicalis was

isolated repeatedlyfrom the blood andurine of

a patient who had open heart surgery. This

strainof C. tropicalisproduced elongated

pseu-dohyphal cells in serum within 3 h and these

weregrossly similar topseudogermtubes. One must recognize the possibility that this may occur more frequently as the identification of yeasts involved in opportunistic infections be-comesmoreprecise.Unfortunately,there is

evi-dence that some laboratories rely exclusively

onthistestfor identificationofC.albicans (14,

20), and thiscannotbejustified. Inouropinion,

apositivepseudogerm tubetest can be

consid-ered presumptive identification ofC. albicans

orC. stellatoidea tobe confirmedbyadditional

tests, whereas a negative test does notexclude

the identificationof these twoyeasts.

Thesequence tobefollowedforidentification ofyeasts isreadilyadaptable foruse inclinical

microbiology(Fig. 9). Sinceprepared disksand

basal media canbe stored, thetests canbeset

uprapidly. Inourexperience, disks storedin a

desiccator have been satisfactory for up to 6

months, with theexceptionofxylose. When the

xylosediskturnsbrown, it is no longerreliable.

Thereissomeadvantage toperformingthe test

for hyphaeorpseudohyphae directly from

sev-eral isolated colonies on theprimary isolation media to determine whether more than one species ispresent. One moreday would be

re-quired, but additional information would be

I

PRODUCTIO OF HYPHAE PSEUDOHY

(1-2days)

SPECIMEN

LABORATORY

ISOLiTED

YEAST COLONIES

MEDIUM FOR HARVESTING

(1-2days)

)N

~~~~~PS

N~~~ -'OR

(PHAE HEAVY

SUSPENSION

RAPID TESTS (1-2 Days)

I

3EUDOGERM TUBETEST (3h)

1. CARBOHYDRATEASSIMILATION

2. NITRATEASSIMILATION

3. FERMENTATION

4.UREASEPRODUCTION

FIG.

9. Sequence of procedures

for

identification

ofyeasts.

obtained in some cases. The inoculum for the

harvesting medium couldbeobtained from the

surface ofthe CMTagar platesince ithas not

been coveredwith a glasscoverslip.

Ourschema isthe same inprinciple asthat

reported by Barnett and Pankhurst (3), i.e.,

identification of asporogenousyeastsbased

pri-marily on physiological tests and multiple en-tries of a species when results of a test are

variable.Theseauthorsalso used the data

pub-lished by Lodder (15) for 341 speciesand added descriptionsof93 newspecies for a total of 434.

Their key to the identification of these yeasts

was constructed after computer analysis

re-vealed the most efficient tests for

differentia-tion among species. Thispublication is

recom-mended for those laboratories interested inan

expanded schema for identification of aspo-rogenous yeasts.

Werecognizethatasearchfor, and

identifica-tionof,yeasts withall specimens is not

practi-cal for clinical laboratories with workloads

al-ready strained. Communication and

coopera-tion between physician and microbiologist is

requiredto identify cases in which recovery of

yeastswouldbearsignificantlyonclinical man-agementofthepatient.Theneedforthis collab-oration has been expressed clearly by Klainer andBeisel(12): "In thissituation, aclose recip-rocal relationship must develop between the clinician atthe bedside and the microbiologist inthelaboratory. Thephysicianmustalert the

microbiologisttothepossibilityofan

opportun-istic infection; in return, the microbiologist

must regard isolation by culture of

'contami-nants' or saprophytes, especially ifrecurring, with caution and suspicion. Together, such a

http://jcm.asm.org/

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34

medical team must attempt to determine the significance of the presence of these orga-nisms."

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1. Adams, E. D.,andB. H. Cooper. 1974.Evaluationof a

modified Wickerham medium foridentifying medi-callyimportantyeasts.Am. J. Med.Technol. 40:377-388.

2. Ahearn, D. G. 1974.Identificationandecology of yeasts of medicalimportance,p.129-146.In J. E. Prierand

H.Friedman (ed.), Opportunistic pathogens.

Univer-sityPark Press,Baltimore.

3. Barnett,J.A.,andR. J.Pankhurst. 1974.Newkey to

theyeasts. AmericanElsevierPress,New York.

4. Buckley,H. R.,and N. van Uden. 1963. The

identifica-tion of Candidaalbicanswithintwo hoursby the use of an egg white slide preparation. Sabouraudia

2:205-208.

5. Dolan,C.T.1971. A practicalapproachtoidentification ofyeast-like organisms.Am. J.Clin. Pathol.

55:580-590.

6. Dolan,C.T., andM. R.Woodward.1971.Identification of Cryptococcusspecies inthe diagnostic laboratory.

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7. Gaines,J.D.,andJ.S.Remington. 1972. Disseminated

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8. Haley,L.D., andP. G.Standard.1973.Techniques for yeastidentification,p. 109-129.InU.S. Department of Health, Education, and Welfare, Center for Disease

Control, Atlanta, Ga.

9. Hutter, R. V. P., andH. S. Collins. 1962. The occur-renceof opportunistic fungus infections ina cancer

hospital. Lab.Invest. 11:1035-1045.

10. Kahanpaa, A. 1972. Bronchopulmonary occurrence of

fungiinadults. ActaPathol. Microbiol. Scand. Sect.

B(Suppl227):1-147.

11. Kauffman, C. A.,andJ. S.Tan. 1974.Torulopsis gla-bratarenalinfection. Am. J. Med. 57:217-224. 12. Klainer,A. S., and W. R. Beisel. 1969. Opportunistic

infection:areview.Am. J.Med. Sci. 258:431-456.

13. Kreger-VanRij, N. J. W. 1969. Taxonomy and

systemat-icsofyeasts, p.5-78.InA. H. RoseandJ.S. Harrison (ed.), The yeasts, vol. 1. Academic Press Inc., New York.

14. Law, E. J., 0. J. Kim, D. D. Strieritz, and B. G.

MacMillan. 1972. Experience withsystemic

candidi-asis intheburned patient.J.Trauma12:543-552. 15. Lodder, J.(ed.).1970. The yeasts.North-Holland

Pub-lishingCo.,Amsterdam.

16. Louria,D.,A.Blevins,D.Armstrong,R.Burdick, and

P.Lieberman. 1967.Fungemia causedby "non-patho-genic"yeasts. Arch. Intern. Med. 119:247-252. 17. Mackenzie, D. W. R. 1964. Morphogenesis of Candida

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18. Mackenzie, D. W. R. 1965.Studiesonthe morphogene-sisof Candida albicans. II. Growth in organ extract.

Sabouraudia4:126-130.

19. MacMillan,B. G., E.J. Law, and I. A. Holder. 1972.

Experiencewith Candida infectionsinthe burn pa-tient. Arch. Surg. 104:509-514.

20. Miller, R. M., S. H.Polakavetz, R.B.Hornick, andR. A. Cowley. 1973. Analysis of infections acquired by theseverely injured patient. Surg.Gynecol. Obstet.

137:7-10.

21. Painter, B. G., andH. D. Isenberg. 1973.Isolation of Candida parapsilosis; report of two cases. Am. J.

Clin. Pathol.59:62-65.

22. Pankey, G. A., and J. R. Dalaniso. 1973. Fungemia causedby Torulopsisglabrata. Medicine52:395-403. 23. Quie,P.G., and R. A. Chilgren. 1971. Acute dissemi-nated and chronic mucocutaneous candidiasis. Semin. Hematol. 8:227-242.

24. Rifkind, D.,T. L.Marchioro, S.A.Schneck, andR. B.

Hill,Jr. p967. Systemicfungal infections complicat-ing renal transplantation and immunosuppressive therapy. Am. J. Med.43:28-38.

25. Silva-Hutner, M., and B. H. Cooper 1974. Medically

importantyeasts, p. 491-507.In E. H. Lennette, E. H.Spaulding, andJ. P.Truant(ed.), Manual of clini-calmicrobiology,2nd ed. AmericanSociety for

Micro-biology,Washington, D.C.

26. Taschdjian, C. L., J. J. Burchall, and P. J. Kozinn. 1960.Rapid identification of Candida albicans by

fila-mentation on serum andserum substitutes. J. Dis.

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27. Webb, C. D., C. Papageorge, and C. T. Hall. 1971.

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28. Wickerham, L.J. 1951. Taxonomyof yeasts. U.S.

De-partment ofAgriculture Technical Bull. 1029. U.S. DepartmentofAgriculture, Washington, D.C.

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