JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1975, p. 206-217Copyright ©) 1975 American Society for Microbiology

Vol. 2, No. 3Printed in U.S.A.

Improved Auxanographic Method for Yeast Assimilations: a

Comparison with Other ApproachesG. A. LAND,* E. C. VINTON, G. B. ADCOCK, AND J. M. HOPKINS

Departments of Clinical Microbiology and Medical Mycology, Wadley Institutes of Molecular Medicine,Dallas, Te-xas 75235

Received for publication 12 May 1975

An improved pour-plate auxanographic method has been developed for deter-mining the assimilation of 14 different carbohydrates by medically importantyeasts. Reduction of a dye incorporated into the agar has been correlated withthe growth and carbohydrate assimilation of the yeasts, allowing the speciationofmany yeasts within 24 to 48 h. This technique has been found to compare morethan favorably with existing yeast assimilation techniques, in terms of rapididentification, total cost, and technician time in preparing and inoculating theplates. The dye pour-plate auxanographic technique provides an easier-to-inter-pret, rapid, and reproducible method of mycological identification for smallclinical laboratories.

In debilitated hosts, such as cancer patients,time spent in the identification of microorga-nisms is of the utmost importance (1, 22, 30).Often the decision between which organism is aprobable pathogen and which is a probable con-taminant becomes critical when deciding uponaddition of antimicrobials to the chemotherapyof these patients. For example, amphotericin B,the antimicrobial of choice for most systemicfungal infections, is not a drug to be promis-cuously given to seriously ill patients, due topossible toxic side effects, especially nephrotox-icity (27). Thus, to prescribe this treatment onthe basis of isolating an unspecified yeast froma patient could further compromise an alreadydebilitated host (29). On the other hand, identi-fication of fungal isolates is notoriously slow,and for this reason, antimicrobial therapy isoften delayed beyond the point where it wouldbe beneficial to the patient in case of a very realinfection.Most techniques for fungal identification in-

volve laboratory demonstration of their mor-phology under various environmental condi-tions, as well as testing for certain biochemicaltraits they might have (2, 15, 21). The latter isparticularly important in speciating medicallyimportant yeasts (1, 30). Identifying organismsin this manner, however, is tedious and usurpsa great deal of technician time. Consequently,most small clinics or hospital laboratories donot take the time to do more than a cursoryidentification, i.e., Candida albicans versusCandida species not albicans (10). There hasbeen, however, an increase in the number ofhospitalized patients with disseminated oppor-

tunistic fungal infections (31) due to alteredhost defense mechanisms (8, 9, 20), surgicalprocedures (26, 29), and manipulation of in-dwelling catheters (35). With this higher inci-dence of opportunistic infections, then, it hasbecome imperative to identify organismsquickly (13, 25) and to quantitate them (22) sothat the time needed for confirmatory cultureswithout endangering the patient is available(G. L. Dorn, G. Burson, and J. Haynes, manu-script in preparation).

The ability of a yeast cell to assimilate var-ious carbohydrates has long been used as thestandard method to speciate medically impor-tant yeasts (7). Since the auxanographic tech-nique of carbohydrate assimilation first ap-peared (7), several methods have been devel-oped to improve the technique and obtain moredefinitive results. Recent advances in yeast as-similative techniques have included a modifiedauxanogram using sugar-impregnated disks(11) and a turbidimetric system in a broth,which has become the standard by which allother techniques are measured (33). Newer mod-ifications of the Wickerham and Burton tech-nique (33) include aeration of the broth (3),solidifying the broth as an agar slant (23), andmodification of the agar slant by the addition ofa pH indicator dye (1).

This latter technique (agar slant and indica-tor dye hereafter referred to as the Adams-Cooper technique) would be attractive to asmall laboratory for several reasons: (i) a goodcorrelation of indicator change with growth,making assimilations easier to determine; (ii)ease of preparation (the carbohydrate-contain-

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AUXANOGRAPHIC METHOD FOR YEAST ASSIMILATIONS 207

ing medium may be autoclaved together inbatch quantities and stored for up to 6 months);and (iii) cost in technician time compared favor-ably with existing methods.

In employing the Adams-Cooper technique(1) of yeast assimilation at Wadley Institutesand Granville C. Morton Hospital, we experi-enced a continual problem ofcontamination (ap-proximately 10 to 15%). Contamination mightbe encouraged in the Adams-Cooper procedure,since the carbohydrate concentration of the me-dium is high and the sterilization procedure hasbeen drastically reduced to avoid hydrolysis ofsome of the more labile carbohydrates.Even though the dye assimilation slant has

achieved a considerable timesaving advantageover other assimilation methods, identificationof medically important yeasts still requires 1 to2 weeks. The first several days ofany procedureused for the identification of yeasts is usuallyconcerned with starving the yeasts (4, 33), therationale for this procedure being to deplete thelarge metabolite pools that are known to occur

in fungi (19). Thus, upon subculture to the com-

plete assimilation medium, the starved cellswould be forced to feed off of the carbohydratesprovided. The Adams-Cooper technique avoidssome time loss as a result of starving theyeasts, by using a very dilute cell suspension asthe inoculum. It takes, however, 2 to 3 days forthis dilute inoculum to grow sufficiently to benoticeable on the slant, which does increaseidentification time. This present study was initi-ated to develop a method of yeast assimilation,based on current technology, but with fasteridentification capabilities. We also felt that anysystem of yeast assimilation should be applica-ble to small clinical laboratories in terms ofcosts, space, technologist time, and ease in iden-tification of the specimen.

MATERIALS AND METHODSMicroorganisms. All yeast isolates came from

the following sources: The Tulane University Mycol-ogy Collection; The Mycology Laboratories, TexasState Department of Health; Department of Microbi-ology, Temple University School of Medicine; De-partment of Microbiology and Immunology, DukeUniversity Medical Center; Clinical MicrobiologyLaboratory, Granville C. Morton Cancer and Re-search Hospital.

Dye pour-plate auxanographic (DPPA) tech-nique. A medium containing a pH indicator dye,which provided the optimum conditions for rapidcarbohydrate assimilation, was determined by vary-ing the pH and concentrations of either the nitrogensource or the carbon source. The composition of themedium supporting the most rapid assimilation ofglucose by both C. albicans and Cryptococcus neofor-mans was as follows (per liter): 20 g of agar (DavisGelatin, Christ Church, New Zealand); 20 mg of

bromocresol purple (Sigma Chemical Company, St.Louis, Mo.); 0.67 g of yeast nitrogen base (YNB;Difco Laboratories, Detroit, Mich.). The above dyeagar medium was brought to a pH of 7 while molten,then dispensed into 4-ounce (0.121 liter) prescriptionbottles (60 ml per bottle) and autoclaved at 15pounds pressure for 15 min. The sterile agar inbottles was used either immediately or after solidifi-cation and storage at 4 C.

The melted medium was allowed to cool to 45 to 50C, seeded with a heavy suspension made from two tothree colonies from a pure culture of the yeast (ap-proximately 107 to 108 yeasts/ml, Table 1), pouredinto plates, and allowed to solidify. After the inocu-lated agar had solidified, carbohydrate-impregnateddisks were placed on the surface of the plates, sixalong the outer perimeter of the plate, approxi-mately 1 inch (2.54 cm) from the rim, and one disk inthe center. The disks were arranged so that the firstseven carbohydrates (see Table 3) were on one plateand the last seven on a second plate.

Carbohydrate disks. All sugars (Table 2) wereobtained from the Sigma Chemical Company (St.Louis, Mo.) and were made up into 100-mg/ml stocksolutions and filter-sterilized (0.20 um Nalge LabWare, Division Sybron Corporation, Rochester,N. Y.). Various concentrations (3 jig to 30 mg) ofthese filter-sterilized stock solutions were dispensedon sterile concentration blanks ('/4-inch [0.635 cm]diameter, Difco Laboratories), placed in sterile plas-tic plates (60 by 15 mm, Kimble Plastics), and al-lowed to dry.

Assimilation slants. Assimilation slants weremade and inoculated according to the procedure ofAdams and Cooper (1). Briefly, the medium con-tained (per 100 ml): 2 g of agar (Noble agar, DifcoLaboratories); 0.2 ml of a 1.6% solution of bromo-cresol purple; 1.0 ml of 0.1 N NaOH, 0.67 g of YNB(Difco Laboratories); 1 g of a specific carbohydrate(Table 2); and 100 ml of deionized water. The me-dium had a final pH of 7.0. The molten medium was

TABLE 1. Evaluation of the influence of size of theinoculum in the rapid identification ofC. albicans by

yeast assimilations

Identifica-Absorbance tion: time

Diion of sNo erg at 600 nm of elapsed fororiginal nisms perinoculum 0.1 m10 the yeast complete

suspension yeast assimi-lations (h)

0 TNb 1.135 171:10 TN 0.185 171:102 TN 0.025 241:103 9707 721:104 88 >96

a Done by averaging replicate spread plate cul-tures of 0.1 ml of each dilution (Sabouraud dextroseagar, incubated at 37 C).

b Too numerous to count on spread plates.c The original suspension contained approxi-

mately 107 organisms/ml.

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208 LAND ET AL.

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dispensed (4.5 ml) into screw-capped tubes (16 by 125mm; Owens-Illinois, Toledo, Ohio), sterilized by au-toclaving at 10 pounds pressure for 10 min, andslanted. Slants were inoculated by pipetting 0.1 mlof a dilute yeast suspension onto the surface of themedium as described by Adams and Cooper (1).Swab auxanographic technique. YNB was pre-

pared in a 10x concentration in 100 ml of deionizedwater according to the manufacturer's directions(Difco Laboratories). The lOx solution was then di-luted with 900 ml of deionized water and 20 g of agar(Davis Gelatin) was added. The medium was auto-claved at 15 pounds pressure for 15 min and allowedto cool to 50 C. Plates (155 by 15 mm, Kimble Plas-tics, Division Owens-Illinois) were poured to a 1/4-inch depth (approximately 60 ml). The plates wereallowed to solidify and were incubated (37 C) over-night to remove moisture. The YNB agar plateswere inoculated from a dilute yeast suspension by amodified swab technique (6). The swab was im-mersed in this yeast suspension and then streakedover the entire surface of the agar several times.Commercially prepared carbohydrate-impregnateddisks (Difco Laboratories; BBL, Cockeysville, Md.)were arranged in the same pattern used for theDPPA technique on two plates. All inoculated platesand slants were incubated at either 37 C or roomtemperature and read at appropriate intervals.Wickerham broth technique. YNB (10x) and car-

bohydrate stock (10%) solutions were prepared asdescribed by Wickerham and Burton (33). The YNBsolution was appropriately diluted, dispensed in 4.5-ml aliquots to screw-capped test tubes, and steri-lized by autoclaving. The carbohydrate stock solu-tions were filter sterilized, and 0.5 ml of each carbo-hydrate was added aseptically to the cooled broth intubes, making the final carbohydrate concentrationin the medium 1%. The carbohydrate-broth tubeswere inoculated as described by Wickerham andBurton, and assimilation results were also judged byhis criteria (33).

Evaluation of techniques. The four yeast assimi-lation techniques were compared with each other fortheir ability to provide rapid assimilation results forknown yeasts. The data collected were the elapsedtime for each carbohydrate used in a particular as-similation technique to become positive and the to-tal time required for the complete assimilation pro-file of the organism.A more detailed comparison of the DPPA and

swab auxanographic techniques was made in thefollowing double-blind study. Specimens sent to theclinical laboratory of Granville C. Morton Cancerand Research Hospital were streaked for isolationon Sabouraud agar plates and incubated at 37 C andat room temperature. Single isolated yeast colonieswere used to inoculate two Sabouraud slants. Theinoculated slants were equally divided between twogroups of technologists for identification. In addi-tion, both groups were provided with known orga-nisms to serve as internal controls for the experi-ment. Both groups followed the same schema foridentification (1), differing only in their method ofyeast assimilation (one group used the DPPA tech-nique and the other group used the swab auxano-

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210 LAND ET AL.

gram). Data were expressed as the total time takenby each group for the identification of known orunknown yeasts.

Growth studies. Five-milliliter suspensions of C.albicans were made as described for the DPPA tech-nique and the absorbance of each suspension wasdetermined at 650 nm. Tenfold dilutions of thesesuspensions were made and their absorbance at 650nm was also determined. One-tenth milliliter ofeach dilution was spread on the surface of a Sabour-aud dextrose agar plate; the process was repeated intriplicate for each dilution. The remaining 4.7 ml ofeach yeast dilution was mixed with molten DPPAagar and allowed to solidify. Carbohydrate-impreg-nated disks were placed on the solidified plates forassimilation auxanography. The numbers of yeastsin each dilution were determined by the aforemen-tioned Sabouraud spread plates and numbers werecorrelated with a specific absorbance value at 650nm, as well as time required for each yeast dilutionto give the complete carbohydrate assimilation pat-tern for C. albicans.A second study involved correlating changes in

absorbance at 600 nm with color change in the bromo-cresol purple dye and with yeast growth. Sealedquartz cuvettes (Gilford Instruments, Oberlin,Ohio) were sterilized and aseptically filled with oneof the following: (i) a molten dye-agar combination,also containing 1.5 mg of glucose and 107 organismsper ml; (ii) the same dye-agar-glucose combinationcontaining 104 organisms per ml; (iii) YNB glucoseagar without dye inoculated with 107 organisms perml; (iv) YNB glucose agar without dye inoculatedwith 104 organisms per ml; (v) a YNB-agar reference

standard containing no dye, glucose, or yeast. Theagar in the cuvettes was allowed to solidify asquickly as possible, without temperature shockingthe yeasts in the suspension. The changes in absorb-ance at 600 nm for each cuvette were monitored andrecorded automatically at 2-min intervals for 24 h.The temperature of the spectrophotometric chamberduring the experiment was 36 ± 1 C.

RESULTSA medium was formulated which would per-

mit the rapid assimilation of carbohydrates byC. albicans as evidence by a pH change frompurple to yellow in an agar pour plate contain-ing bromocresol purple (Table 1). Further-more, it was our experience that a visible (pur-ple to yellow) color change was apparent 2 to 4 hafter inoculation with not only glucose but withseveral of the other carbohydrate substrates aswell (Fig. 1B and C). By varying concentrationsof each of the carbohydrates (Table 2), we deter-mined the optimal concentrations for theirrapid assimilation via the DPPA technique (ex-pressed as milligrams/disk): (i) glucose, galac-tose, sucrose, and lactose, 3 to 6 mg; (ii) sorbose,maltose, and dulcitol, 5 to 7 mg; (iii) cellobiose,10 to 14 mg; (iv) trehalose, raffinose, melizitose,and xylose, 10 to 12 mg; (v) melibiose, 15 to 20mg; (vi) inositol, 2 to 4 mg.The rapid assimilation of carbohydrates by

C. albicans also appeared to be in direct propor-

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E4 lI ~~~ ~~~~~~~~~~F4 1FIG. 1. The assimilation of various carbohydrates by C. albicans using an improved DPPA technique: (A)

1 h, (B) 3 h, (C) 5 h, (D) 10 h, (E) 18 h, (F) 24 h. Numbers correspond to the carbohydrates listed in Table 2.

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AUXANOGRAPHIC METHOD FOR YEAST ASSIMILATIONS

tion to the size of the inoculum (Table 1). Re-sults obtained from plating 10-fold dilutions of a5-ml yeast suspension for enumeration as wellas carbohydrate assimilation indicated that anabsorbance of 0.2 to 1.1 at 600 nm, which corre-lated with 106 to 107 yeasts per ml in the suspen-sion, gave a complete carbohydrate assimila-tion profile within 17 h (Fig. 1). Ifthe concentra-tion of yeasts in the suspension was increased10-fold, i.e., from 107 to 108 organisms per ml,the time for a complete assimilation patternwas reduced to around 12 to 14 h.

There also appeared to be a correlation be-tween size of the inoculum, growth, and achange in color of the dyed agar from purple toyellow (Fig. 2). When the DPPA agar in acuvette was inoculated with 105 organisms perml, a steady decrease in absorbance at 600 nmwas noted through 18 h, whereas during thissame time a parallel cuvette containing yeastagar suspension without dye increased in ab-sorbancy at 600 nm. Upon direct observation,the dye agar yeast suspension was completelyyellow by h 14. Continuous spectrophotometricmonitoring showed that the rates of change ofabsorbance at 600 nm for the growth cuvette(YNB plus yeasts) and dye reduction cuvette(dye plus YNB plus yeasts) were approximatelythe same through 12 h (Fig. 2); i.e., time of

ioaa

maximum growth correlated with the time ofmaximum dye reduction. Changes in growthand dye reduction continued until h 20, where-upon a stationary and parallel phase for bothwas noted. Dye agar inoculated with 107 orga-nisms per ml showed an immediate changefrom purple to yellow (approximately 1 h) and acompanion culture without dye showed a slightincrease in turbidity at 600 nm during h 1. Bydirect observation at h 4, dye reduction wasapparently completed. The cuvette containingyeasts plus YNB showed a modest increase inturbidity by this time; and, by h 4, the absorb-ance of both cuvettes at 600 nm was approxi-mately the same. By h 8, both cuvettes showeda very limited rate of change and the rates ofabsorbance of both cuvettes paralleled eachother.

In determining the rate of carbohydrate as-similations by known yeasts with either theDPPA technique or with the assimilation slant,swab auxanogram, or Wickerham broth, theDPPA technique consistently gave more rapidand easier-to-read results (Fig. 3, Table 3). Theaverage time for the complete assimilation pro-files of all yeasts in this study was as follows:DPPA, 1.4 days; assimilation slant, 10.9 days;swab auxanogram, 13.4 days; Wickerhambroth, 16.7 days. The data broken down intogeneric groups show the same trends in rapid-ity of carbohydrate assimilation techniques;i.e., DPPA results were faster than assimila-tion slant and swab auxanogram, with Wicker-ham broth the slowest of the four. The genera ofCandida and Rhodotorula as groups were allspeciated within 1 day by the DPPA technique,as was Torulopsis glabrata and Trichosporoncutaneum. Final results with the assimilationslants for these fungi took 7 to 10 days, theswab plates 11 to 14 days, and the Wickerhambroth greater than 14. Species of Cryptococcus,although following the same declension in time

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FIG. 3. The auxanographic assimilation of var-

ious carbohydrates on DPPA by Cryptococcus ncofor-mans. Numbers correspond to the carbohydrateslisted in Table 2.

TIME XFORA)

FIG. 2. Correlation of dye reduction with growthduring pour-plate auxanography. Orgslml, Numberoforganisms in 1 ml of inoculum; BCP, bromocresolpurple.

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212 LAND ET AL.

for complete yeast assimilation on the variousagars as the other fungi had, were slower as agroup in giving complete assimilation patterns.For example, the Cryptococcus group averaged2 days for complete assimilation patterns on theDPPA. The other carbohydrate assimilation

techniques also required correspondinglylonger time to give complete assimilation re-sults.The comparison of the DPPA with the swab

technique in the identification of yeasts iso-lated from clinical material also showed that

TABLE 3. Comparison of the DPPA technique ofyeast assimilation with a swab auxanographic (SA)technique in identifying either known yeasts or unknown yeasts isolated from patient material

OrgnmNo. of Days required for complete assimilationOrganism Medium isolatesisolates 1 2 3 >3

Candida albicans DPPA 56 88a 100

C. aaseri

C. utilis

C. tropicalis

SA

DPPASA

DPPASA

DPPASA

56

11

lblb

1111

100

86 100

100

100

73 100

100

55 100

C. stellatoidea DPPASA

C. parasilosis

C. krusei

C. guilliermondii

C. pseudotropicalis

Cryptococcus neoformans

C. albidus var. albidus

C. albidus var. laurentii

C. uniguttulatus

C. terreus

C. luteolus

Rhodotorula glutinis

R. graminis

DPPASA

DPPASA

DPPASA

DPPASA

DPPASA

DPPASA

DPPASA

DPPASA

DPPASA

DPPASA

DPPASA

DPPASA

2222

12"

22

1010

1414

2222

55

44

11

3b3b

11

11

22

92 100

55 100

10050

60 80 10060

71 94 10057

59 77 10068

20 100

100

77 100

50 100

100

100

100

100

100

75 100100

100

33

100

50 100

33

100

100

100

100100

100

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AUXANOGRAPHIC METHOD FOR YEAST ASSIMILATIONS 213

TABLE 3-Continued

No. of Days required for complete assimilationOrganism Medium isolatesisolates 1 2 3 >3

R. rubra DPPASA

11

100100

Sporobolomyces salmonicolor

S. roseus

DPPASA

DPPASA

Saccharomyces cerevisiae DPPASA

44

25 75 10025 100

11

1919

100

42 79 10021 52

S. champagnii

S. rouxii

100100

Torulopsis glabrata

Trichosporon cutaneum

T. pullulan

Aureobasidium pullulan

A. prunorum

DPPASA

DPPASA

DPPASA

DPPASA

DPPASA

2626

77

50 66 10038 69

100

11

57 100

100

10022

lb100

Geotrichum candidum

Ustilago zeae

DPPASA

22

50 100

DPPASA

100

50 100100

a Data expressed as percentage identified within a specified time period.bCultures provided from either the Tulane University Mycology Collection or the mycology laboratories,

Texas State Department of Health.

the DPPA approach gave a faster assimilationprofile (Table 3). This was a double-blind study,with two separate groups of technologists isolat-ing and identifying yeasts from either sputum,biopsies, autopsy tissue, or blood, in addition tosome known fungal cultures for referencestrains. The DPPA group averaged 95% identifi-cation by 3 days and 100% identification by 4days. The swab plate group averaged 45% iden-tification of all fungi by 3 days, 65% by day 7,and 100% by day 13. The complete data fromboth groups showed a 90% correlation on identi-fication (only 243 of the 300 strains or isolates

identified are reported in this paper). Resultsfrom this study also demonstrated that pureyeast colonies obtained from various primaryisolation media (i.e., Sabouraud dextrose agar,eosin methylene blue agar, blood agar plates,corn meal plus Tween 80, rice extract plusTween 80, and Salmonella-Shigella agar) couldbe made into suspensions and be used for theDPPA technique without a prior period ofgrowth and starvation before their assimilationpattern was determined. However, the swabplates tended to show some background growthwhen prepared directly from an isolation plate.

DPPASA

33

100

100

DPPASA

22

100100

100

100

100

100

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214 LAND ET AL.

DISCUSSIONThe increased incidence of opportunistic fun-

gal infections in patients undergoing treat-ments with corticosteroids (24, 25), surgical ma-nipulations (26, 29), catheterization (35), immu-nosuppression (17, 22), or in the advanced stateof malignancy (8, 9, 14, 18, 31) has made itnecessary for clinics and hospitals to improvetheir fungal identification techniques. Mostsmall clinics and laboratories, however, havenot taken the time in the past for proper fungalidentification, for reasons of work overload andlack of technical expertise (1, 10). Althoughmany techniques and media have been devel-oped to streamline the identification of bacteriafrom clinical samples, particularly the Entero-bacteriaceae (16), few techniques have been de-veloped to provide the same service in identify-ing fungi.

Traditionally, yeasts have been identified bycharacteristic fermentation (gas production) ina carbohydrate-containing broth and by growthwithout gas production in a carbohydrate-con-taining broth, i.e., assimilation (1, 4, 21, 33).Yeast carbohydrate assimilations done in thebroth system, as well as in a solidified version(23), require some type of starvation procedure(4, 33) prior to inoculation into a medium con-taining the specific carbohydrate. The starva-tion procedure was deemed necessary to de-crease background growth, made possible bycarbohydrate pools within the yeast, whichcould give false positive growth on latent orweakly assimilated substrates. The swab platetechnique provided an example of backgroundgrowth during the present study if the plateswere seeded directly with cells grown on a richmedium, but not ifthe inoculum had been previ-ously starved. However, the starvation proce-dure increases the time required for yeast iden-tification.A recent modification ofthe standard method-

ology for assimilations involved the addition ofa dye (bromocresol purple) to a solidified Wick-erham medium (1). Results obtained with thistechnique suggested that a change in color frompurple to yellow could be correlated with thegrowth of the yeast on the substrate. Althoughthe dye-slant technique for yeast assimilationprovides faster, easier-to-interpret, and morereproducible results, there are some inherentproblems with the procedure. One disadvan-tage is that several days are lost as the diluteinoculum on the slant grows enough to be no-ticed by the technologist. Another problem isthat with a multitude of tubes (13 to 15 depend-ing upon the number of sugars used) for eachtest, and with several yeasts per day to iden-

tify, the technologist could easily lose track ofwhich tube has or has not been inoculated. Afinal disadvantage, at least in our experience,is a consistent 10 to 15% contamination rate inthe uninoculated assimilation slants.The evolution of the DPPA technique was, as

stated in the introduction, to provide a rapid,more reproducible, and less sophisticatedmethod of yeast assimilation (1, 10). The use ofa dye-agar auxanogram was also explored byAdams and Cooper (1); and the auxanogramscompared favorably with their slants. How-ever, they used a swab technique to inoculatethe plate which might tend to give ambiguousresults for weak or latent assimilations of carbo-hydrates. The original auxanographic tech-nique was a pour plate (7); we felt that thepour plate would also provide a tightly definedring of growth around the substrate-impreg-nated disk, since the size of the ring would becontrolled by the diffusion of substrate from thedisk into the agar.Dye was added to the agar based on the sug-

gestion of Adams and Cooper (personal commu-nication) that growth seemed to be correlatedwith a pH change in the medium (1), with thebromocresol purple indicator changing frompurple to yellow. Studies presented in this pa-per confirm that observation, for both a highinoculum and a low inoculum showed dye reduc-tion and a concomitant increase in turbidity;the latter observation has classically been inter-preted as growth. Admittedly, the changes inturbidity of either inoculum was not striking,but then cellular division would not be expectedto occur at a rapid rate with a large inoculum inthe microaerophilic environment of a pourplate. However, in comparing results from theturbidity experiments and the dilution plateswith regard to time needed for the as imilationpattern of a yeast to be completed, our resultssuggest that dye reduction occurs rapidly in thepresence of 106 to 108 cells per ml and has unfail-ingly correlated with the assimilation of a par-ticular carbohydrate.The nitrogen source also affected the rate of

carbohydrate assimilation. For example, con-centrations of YNB suggested by the manufac-turer to be optimal for growth appeared tocause a considerable delay in the production ofacid from carbohydrate metabolism. Decreas-ing the concentration of YNB to 0.1 or 0.01times the suggested concentration permitted amore rapid appearance of a change in pH indica-tor to yellow. There are several good reasonswhy the limitation of nitrogen could affect car-bohydrate assimilation. For instance, the nitro-gen source could affect the metabolism of the

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AUXANOGRAPHIC METHOD FOR YEAST ASSIMILATIONS

cell: first of all, limited nitrogen would createstarvation conditions within the cells after sev-

eral metabolic turnovers, especially if theyeasts are inoculated in high numbers. In addi-tion, a decrease in YNB would mean a concomi-tant decrease in the amount of NH4, ion in themedium, as ammonium sulfate is one of themedium's constituents (0.5%). A decrease in theNH4, ion has been shown to affect carbohydratetransport in yeast and other fungi as well as

influencing their carbohydrate metabolism byaffecting some key enzymes for control of glycol-ysis and energy production (reviewed in 5, 12,32, 38).

There are some disadvantages to the DPPAtechnique. The pour plate itself is felt by some

to be an extremely messy technique and more

open to contamination. Although the latter istrue, the large inoculum used in this methodwould in most cases favor overgrowth of theplate by the inoculated organisms rather thanby chance airborne or shed contaminants, nor

have we been bothered with undue contamina-tion since employing the DPPA technique inour laboratory. The heavy yeast suspensionused as an inoculum requires several colonies,which may be a disadvantage if the sample islimited. However, most primary plates haveenough growth for the test to be done ade-quately and current research indicates that theDPPA technique may be adapted to severalmicro-techniques using one colony as an inocu-lum (Land, Hopkins, and W. H. Fleming III,manuscript in preparation). Temperature con-

trol of the molten agar is also critical, and toavoid mistakes requires a water bath specif-ically set for that one temperature. If the agar

is too hot (above 50 C), labile organisms suchas some of the Cryptococcus species will be heat-killed to a level that would make rapid andcorrect interpretations of the plate difficult. Ifthe agar is too cool (below 45 C), the agar willbegin to gel, giving a very heterogenous suspen-sion and plate, again making interpretation ofresults difficult. The use of two plates may alsobe a disadvantage, but it has been our experi-ence that two plates and seven sugars per plateproduce the easiest results to read and the mostcomplete information. On occasion, we haveused one plate and 14 sugars, and it proveda bit crowded and required some experience inreading results, especially if sugars were

weakly or latently assimilated.We do feel that the advantages of this tech-

nique outweigh the disadvantages. For exam-

ple, the use of the DPPA technique with 14sugars can rapidly speciate 90% or better of thecommon yeasts a clinical laboratory will re-

ceive, without having to do the time-consumingfermentations. The shelf life in prescription bot-tles is at least 6 months. The DPPA may beinoculated from a primary plate without starva-tion, thus decreasing total identification time.The composition of the medium as well as theinoculum have also been formulated to giverapid assimilation results, and in the case ofmost medically important yeasts, identificationwithin 24 h is not uncommon. Organisms whichgrow at 37 C may be tested at that temperature,in most cases achieving more rapid yeast as-

similation profiles than at room temperature.A cost comparison of the DPPA and the assimi-lation slant (Table 4) shows that they both are

comparable. Admittedly, cost comparisons are

TABLE 4. Comparison of cost and time per test in preparing the dye auxanogram plates and the dyeassimilation slants

Auxanogram Assimilation slants

Item Cost Item Cost

Petri plates ....................... 0.46 TubeSa.3.45YNB ............................ 0.01 YNB.0.04Davis agar ........................ 0.03 Noble agar.0.094-oz prescription bottlesa ...... ..... 0.21Bromocresol purple ................ 0.01 Bromocresol purple.0.005Technician timeb Technician timeb

Preparation and autoclaving of me- Preparation and autoclaving of me-dium .......................... 10.50 dium.18.00Labeling and preparation of suspen- Labeling and preparation ofsuspen-sions ............................ 2.00 sions.5.00Inoculation of medium for 20 tests 3.20 Inoculation of medium for 20 tests 5.20

Cost/test .......................... 0.82 Cost/test.1.59

aItems autoclavable and reusable.b Medium for 20 tests and evaluated at $4.00 per h.

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somewhat artificial and the disparity of cost pertest between the two techniques probably is notthat striking.

However, the ease of preparation, ease ofinoculation, the saving of space, and the quick,reproducible, and easy-to-interpret results ofthe DPPA suggest that this technique might beconsidered by a small laboratory for yeast as-

similations. It certainly fulfills the require-ments (1, 10) for a less sophisticated and more

timesaving method of mycological identifica-tion for use in clinical laboratories. Thus, a

laboratory using an easy-to-follow schema ofgeneric identification, such as proposed bySilva-Hutner and Cooper (30), and the DPPAtechnique to speciate the organisms could pro-

vide an adequate mycological identification ser-

vice with a minimum expenditure of time andmoney.

ACKNOWLEDGMENTSWe express our gratitude to B. H. Cooper for helpful

criticisms and suggestions for this study and in the prepara-tion of this manuscript. Additional thanks are given to N.F. Conant, L. Friedman, H. Gallis, J. D. Schneidau, and J.Steadham for the generous donation of cultures for theseexperiments. The technical assistance of S. Chabot, B. Kil-roy, P. Russ, and D. Scott is also acknowledged and appreci-ated.

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