JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1976, p. 186-190Copyright © 1976 American Society for Microbiology

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

Rapid in Vitro Conversion and Identification ofCoccidioides immitis1

S. H. SUN, M. HUPPERT,* AND K. R. VUKOVICHMycology Research Laboratory, Verterans Administration Hospital, Long Beach, California 90801;* and

Department of Medical Microbiology, University of California, Irvine, California 90822

Received for publication 15 October 1975

Since many contaminating nonpathogenic fungi resemble Coccidioides immi-tis culturally, isolates from patients must be inoculated into laboratory animalsto confirm identification as C. immitis. This procedure is time-consuming,expensive, and not generally available in clinical laboratories. When cultures ofC. immitis were grown in slide cultures on modified Converse liquid medium inpurified agar and incubated at 40 C in a candle jar, all 57 isolates demonstratedinhibition of mycelial growth and conversion of arthrospores to endosporulatingspherules in 3 to 5 days. Representative isolates of six species of nonpathogenicfungi that resembled C. immitis culturally either did not grow or failed toproduce endosporulating spherules under the same conditions. This procedure isrecommended for confirming the identification of a culture as C. immitis.

Coccidioidomycosis is a pulmonary diseasecommon in arid regions of the western hemi-sphere. The prevalence of infection in the popu-lation frequently exceeds 50% (5) and approxi-mately one-half of those infected develop symp-tomatic disease (11). Definitive diagnosis is es-tablished by isolating the fungus, Coccidioidesimmitis,or by visualization of characteristic en-dosporulating spherules in pathological speci-mens. Identification of cultures involves thedemonstration of arthrospores and reproduc-tion of the disease in laboratory animals. Thelatter requirement is not academic since manynonpathogenic fungi resemble C. immitis incultural and morphological characteristics (6),and a significant number (15 to 20%) of isolatesof C. immitis produce atypical cultures (8). Re-production of the disease in laboratory animalsis expensive, inconvenient, and time-consum-ing (1 to 3 weeks), and, in addition, many lab-oratories are not equipped to maintain small-animal colonies.A method more efficient and convenient than

animal inoculation would be desirable. Con-verse (3, 4) described a chemically defined liq-uid medium in which mycelial phase growth ofC. immitis would convert reliably to endosporu-lating spherules. This required continuousshake-culturing at 40 C with increased CO2tension. Roberts et al. (12), using a modifiedConverse liquid medium and stationary cul-tures, reported successful conversion to endo-sporulating spherules with 201 isolates of C.

I Veterans Administration project no. 0641-01.

immitis. This would obviate the need for ani-mal infection, but the method has not beenconsistently successful in our experience.Brosbe (2) incorporated a modified Converseliquid medium (9) into agar and reported invitro development of endosporulating spheruleswith four isolates of C. immitis. This was ac-complished in small petri dishes (50 by 12 mm)with incubation at 40 C and in a gas mixture of20% C02-80% air. The Brosbe method should beuseful in clinical laboratories. This report con-cerns confirmation of his findings and modifica-tions of the procedure to make it readily adapt-able for use in clinical laboratories.

MATERIALS AND METHODSCultures. All 57 isolates of C. immitis used in this

study were recovered from human cases of coccidioi-domycosis. Four of these were primary isolates re-isolated from inoculated mice, and the remainderhad been transferred in Sabouraud dextrose agar forseveral generations. Some of these were atypical asdescribed earlier (8). In addition to the proven cul-tures ofC. immitis, one isolate of each ofthe followingfungi that resemble C. immitis was included: Geotri-chum sp., Trichosporon cutaneum, Endomycopsischodati, Myxotrichum sp., Auxarthron reticulatus,and Pseudoarachniotus sp.

Media. All cultures were grown on glucose-yeastextract medium containing 1% glucose, 0.5% yeastextract, and 1.5% agar. Cultures were incubated atroom temperature until arthrospores were present.Converse liquid medium, modified according to theprocedure of Levine et al. (9) was used as the sus-pending liquid for arthrospores and also incorpo-rated into 1% Ionagar no. 2 (MCM-A) for conversionof arthrospores to endosporulating spherules (2). A

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RAPID IDENTIFICATION OF C. IMMITIS

layer of water agar (1.5%) was used in chambers as asource of moisture for incubating slide cultures. Allmedia were sterilized by autoclaving.

Slide culture procedure. A small block of agar(approximately 10 by 10 by 3 mm) was cut from apoured plate of MCM-A and transferred to a sterilemicroscope slide in a sterile petri dish. The agarblock was inoculated on the upper surface with aloop of arthrospore suspension prepared by adding adrop of Converse liquid medium, as modified byLevine et al. (9), to the top of a culture on glucose-yeast extract medium, allowing the spores to floatup, and picking them up with the loop. A sterileglass cover slip was placed on top of the inoculatedblock of agar and pressed gently to secure it to theagar and to flatten the agar slightly. In separateexperiments, the slides were placed either in ananerobic incubator (National Appliance Co., model3640-5) or in a candle jar. When the anaerobic incu-bator was used, slide cultures were placed in a moistchamber (a petri dish, 150 by 15 mm) containing alayer of water agar and two applicator sticks tosupport the slides (maximum of four slides per moistchamber). Dishes were placed in the anaerobic incu-bator and incubated at either 37 or 40 C with a gasmixture of 20% C02-80% air. A small cylindrical jar(18 cm high by 12 cm in diameter) was used as acandle jar. A double layer of filter paper was placedin the bottom and saturated with sterile water tosupply moisture. Slide cultures were placed in anupended rectangular glass slide holder (from astaining jar) so that slides remained horizontal. Thiswas placed in the cylindrical jar and a small candlestanding in a small glass petri dish was placed ontop of the slide holder. A thin roll of modeling claywas attached to the rim of the cylindrical jar. Afterthe candle was lighted, a glass petri dish cover wasplaced on top of the cylindrical jar and sufficientpressure was applied to seal the unit. The entireunit was placed in an incubator either at 37 or 40 C.Slide cultures were observed at zero time and every24 h for development of endosporulating spherules.Photomicrography was accomplished with a NikonMicroflex model AFM microscope at x600 magnifi-cation with Kodak Royal Pan film (10.2 by 12.7 cm).

RESULTSThe sequence of morphological changes un-

der different experimental conditions wereidentical for all of the cultures of C. immitistested. In addition, the sequence of morphologi-cal events was essentially the same for all ofthenonpathogenic fungi (only minor and nonsigni-ficant differences). Therefore, only typical ex-amples will be described.

C. immitis. Arthrospore suspensions at zerotime contained single spores, chains of severalspores, and very few hyphal strands (Fig. la).When incubated at room temperature, germi-nation and hyphae were apparent at 24 h (Fig.lb) with branching prominent after 48 h. Mostisolates showed typical arthrospore formationin 4 to 5 days (Fig. lc). When incubated at 37 C

in either the anaerobic incubator or the candlejar, endosporulating spherules were present in4 to 5 days, but the slide culture was overgrownwith hyphae and many newly formed arthro-spores had developed. Morphological changeswere identical when slide cultures were incu-bated at 40 C in both the anaerobic incubatorand the candle jar. Arthrospores had becomeround cells at 24 h (Fig. ld), and these hadenlarged and their contents appeared granularat 48 h (Fig. le). Endosporulating spheruleswere obvious in 4 to 5 days (Fig. lf). Hyphalformation was markedly inhibited at 40 C andno newly formed arthrospores were apparent.Nonpathogenic fungi. Suspensions at zero

time contained arthrospores, singly and inchains, as well as short strands of hyphae (Fig.2a). Continuous incubation at room tempera-ture resulted in germination, formation of hy-phae, and development of new arthrospores in 4to 5 days (Fig. 2b). A few of these cultures couldbe easily confused with C. immitis by inexperi-enced personnel. Some species did not grow at37 or 40 C, and the remainder developed simi-larly in both the anaerobic incubator and can-dle jar. Round cells were formed at 24 h (Fig.2c). These either degenerated in 4 to 5 days(Fig. 2d) or germinated into hyphae with chla-mydospores (Fig. 2e) and newly formed arthro-spores (Fig. 2f). None ever formed endosporu-lating spherules.

DISCUSSION

Three primary questions were considered inthis study. Would all isolates of C. immitisdevelop endosporulating spherules on theMCM-A medium? Would fungus species thatresembled C. immitis fail to do this? What werethe optimal conditions for consistent morpho-genesis to endosporulating spherules and con-venient use in clinical laboratories?

All 57 isolates of C. immitis converted readilyfrom arthrospores to endosporulating spherulesbut none of the nonpathogenic species did so.Some of the latter did produce large round cellscomparable in size to spherules and, therefore,it is absolutely necessary that an unknown iso-lant be observed to produce and release endo-spores (Fig. lf) before it is identified as C.immitis. This was detectable in some isolatesby 3 days and in almost all by 4 days. Roberts etal. (12) reported 95% conversion by 3 days withtheir liquid culture method, so our method isapproximately 1 day slower. Significantly,however, we found that the most recent iso-lants developed endosporulating spherules themost rapidly.The culture medium and environmental con-

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188 SUN, HUPPERT, AND VUKOVICH

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CX fFIG. 1. Morphological stages in the development of C. immitis: (a) the arthrospore inoculum; (b) arthro-

spores germinating after 24 h at room temperature; (c) typical new arthrospore formation after 4 to 5 days atroom temperature; (d) round cells formed from arthrospores after 24 h of incubation at 40 C with 20% CO2;(e) enlarged round cells with cleavage planes after 48 to 72 h of incubation at 40 C with 20% CO2; (/9endosporulating spherules after 4 to 5 days at 40 C with 20% CO2.

ditions used in this study resulted in consistent temperature, spherulation occurred readily andconversion of arthrospores to endosporulating mycelial formation was inhibited (see also ref-spherules. Although this occurred at both incu- erences 1, 4). Mycelia developed regularly dur-bation temperatures, there were distinct ad- ing incubation at 37 C and, in addition, arthro-vantages favoring incubation at 40 C. At this spores were formed. These represent a definite

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RAPID IDENTIFICATION OF C. IMMITIS

hazard for infection of laboratory personnelwhen slide cultures are removed from the incu-bation chamber for microscope observation.

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FIG. 2. Representative morphological stages in the development of nonpathogenic fungi: (a) the arthro-

spore inoculum; (b) new crop of arthrospores after 5 days at room temperature; (c) round cells formed from

arthrospores after 24 h at 40 C with 20% C0,; (d) degenerating round cells after 5 days at 40 C with 20%

C02; (e) formation of hyphae and chlamydospores after 5 days at 40 C with 20% CO,; (/9 germinating round

cells, hyphae, and new crop of arthrospores after 5 days at 40 C with 20% CO,. The fungi illustrated are A.

umbrinum (a), A. reticulatus (b, /9, Pseudoarachniotus sp. (c, d), and Myxotrichum sp. (e).

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190 SUN, HUPPERT, AND VUKOVICH

culturing demonstrated that all cultures werekilled by this treatment. This procedure is in-convenient, however, because only a single in-cubation time can be used or replicate slidecultures must be prepared for each isolate.Therefore, incubation at 40 C is recommended.The increased CO2 tension has been shown byseveral investigators to favor development ofspherules (1, 10). In this regard, the simplecandle jar arrangement proved as effective asthe controlled gas mixture.

In our opinion, the slide culture method ismore convenient than the liquid culture proce-dure described by Roberts et al. (12). Liquidcultures required the use of 50-ml Erlenmeyerflasks, preliminary observation through thebottom of the flask on the stage of an invertedmicroscope to determine that large round cellshad formed, and preparation of slides to estab-lish that endosporulating spherules were pres-ent. In contrast, a number of slide cultures canbe tested simultaneously in a single candle jarand the slides can be removed daily for micros-copy and returned to the candle jar for contin-ued incubation if spherulation has not oc-curred. Both methods require an incubator setat 40 C, but only a small one is needed for theslide culture method. Although we believe thatslide cultures incubated at 40 C do not repre-sent a potential for laboratory infection, somepersonnel may be uncomfortable with handlingsuch preparations. If so, the original Brosbemethod can be used; i.e., MCM-A in small petridishes and incubation in a candle jar at 40 C.These dishes can be observed daily with low-power objectives without opening the lids and,when large round cells are seen, slides can bemade to determine whether endosporulatingspherules are present. This would be an ap-proach similar to that of Roberts et al.: it offersthe advantage of smaller culture containers butlacks the convenience of slide cultures.

Finally, one should be aware that the use ofrefined agar yields better results than the usualbacteriological agar. Brosbe reported that Iona-gar no. 2 (Colab Laboratories, Inc.), purifiedagar (Difco), and agarose (Bausch and Lomb,Inc.) supported conversion of arthrospores toendosporulating spherules equally well. Ourexperience has been similar and, in addition,we have evaluated these three agars with re-spect to formation of endosporulating spher-ules. They are rated in descending order ofefficiency as follows: Ionagar no. 2, agarose,

purified agar.The conditions that are necessary to induce

morphogenesis of arthrospores to endosporulat-ing spherules are relatively simple. These con-sist of the culture medium (MCM-A) and incu-bation at 40 C in an atmosphere with increasedCO2 tension. The temperature in the incubatoraround the cultures should be checked. If itexceeds 41 C, little or no growth will result.The agar should be inoculated with a sporesuspension sufficiently dense to ensure ease oflocating cells undergoing morphogenesis; thedenser the suspension, the better the conver-sion. In our experience, more than 90% of thearthrospores will convert to endosporulatingspherules. Furthermore, since completingthese studies, we have taught this method totwo persons from other laboratories and theyhave achieved successful conversions.

LITERATURE CITED

1. Breslau, A. M., and M. Kubota. 1964. Continuous invitro cultivation of spherules of Coccidioides immitis.J. Bacteriol. 87:468-472.

2. Brosbe, E. A. 1967. Use of refined agar for the in vitropropagation of the spherule phase of Coccidioidesimmitis. J. Bacteriol. 93:497-498.

3. Converse, J. L. 1955. Growth of spherules of Cocci-dioides immitis in a chemically defined liquid me-dium. Proc. Soc. Exp. Biol. Med. 90:709-711.

4. Converse, J. L. 1956. Effect of physico-chemical envi-ronment on spherulation of Coccidioides immitis in achemically defined medium. J. Bacteriol. 72:784-792.

5. Edwards, P. Q., and C. E. Palmer. 1957. Prevalence ofsensitivity to coccidioidin with special reference tospecific and nonspecific reactions of coccidioidin andhistoplasmin. Dis. Chest 31:35-60.

6. Emmons, C. W. 1967. Fungi which resemble Cocci-dioides immitis, p. 333-337. In L. Ajello (ed.), Coccidi-oidomycosis. University of Arizona Press, Tucson.

7. Friedman, L. D., D. Pappagianis, R. J. Berman, and C.E. Smith. 1953. Studies on Coccidioides immitis, mor-phology and sporulation capacity of forty-sevenstrains. J. Lab. Clin. Med. 42:438-444.

8. Huppert, M., S. H. Sun, and J. W. Bailey. 1967. Natu-ral variability in Coccidioides immitis, p. 323-328. InL. Ajello (ed.), Coccidioidomycosis. University of Ari-zona Press, Tucson.

9. Levine, H. B., J. M. Cobb, and C. E. Smith. 1960.Immunity to coccidioidomycosis induced in mice bypurified spherule, arthrospore, and mycelial vac-cines. Trans. N.Y. Acad. Si. 22:436-449.

10. Lones, G. W., and C. L. Peacock. 1960. Role of carbondioxide in the dimorphism of Coccidioides immitis. J.Bacteriol. 79:308-309.

11. Pappagianis, D. 1967. Epidemiological aspects of respi-ratory mycotic infections. Bacteriol. Rev. 31:25-34.

12. Roberts, J. A., J. M. Counts, and H. G. Crecelius. 1970.Production in vitro of Coccidioides immitis spherulesand endospores as a diagnostic aid. Am. Rev. Resp.Dis. 102:811-813.

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ERRATAEffect of Types of Media on the Production of Acid from

Glucose by So-Called Glucose-Negative Strains of Neisseriagonorrhoeae

ELLEN S. BARON AND ARTHUR K. SAZ*

Georgetown University, Department of Microbiology, Schools of Medicine and Dentistry,Washington, D.C., 20007

Volume 3, no. 3, p. 333, reference 11: Change to "Cultivation properties ofNeisseria gonorrhoeaegrown in chemically defined media. Can. J. Microbiol. 18:1087-1090."

Rapid in Vitro Conversion and Identification ofCoccidioides immitis

S. H. SUN, M. HUPPERT,* AND K. R. VUKOVICHMycology Research Laboratory, Veterans Administration Hospital, Long Beach, California 90801*; and

Department of Medical Microbiology, University of California, Irvine, California 90822

Volume 3, no. 2, p. 186, paragraph beginning with "Cultures," line 11: Change "reticulatus" to'umbrinum."Volume 3, no. 2, p. 189, legend for Fig. 2: Change "reticulatus" to "umbrinum."

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