Simple and Reliable ssimilation Test for the Identification of Candida Species MRION V. MRTIN, M.D., ND J. D. SCHNEIDU, JR., PH.D. Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana 70112 BSTRCT Martin, Marion V., and Schneidau, J. D., Jr.: simple and reliable assimilation test for the identification of Candida species. mer. J. Clin. Path. 53: 875 879, 1970. relatively simple, reliable, and easily readable assimilation test is described. The usefulness of assimilation vs. fermentation patterns for identification was determined for the seven strains of Candida most frequently isolated from clinical specimens. C. albicans,, C. krusei, and C. guilliermondii could be identified easily by means of the assimilation test alone. C. albicans and C. parakrusei had identical assimilation patterns and required either chlamydospore production or fermentation tests for differentiation. C. tropicalis could be identified readily by sugar fermentation alone. The ability to assimilate cellobiose is peculiar to the latter species, but is not found in all strains. THERE are several means of identifying yeasts for taxonomic and diagnostic purposes. Various workers have described rapid means of identifying Candida albicans, one of the most frequently encountered yeasts, but these methods are not 100% reliable, due to the fact that other organisms, notably, and rarely, C. utilis, C. rugosa and Schizosacch.arom.yces fragilis behave like C. albicans on cornmeal agar, in serum and serum substitutes, and in sugar fermentation. 3 ' 4 T - 8 " 10 Carbon assimilation tests have been used for many decades in the classification and Received September 12, 1969; accepted for publication October 29, 1969. Supported by Public Health Service Grant R01 I-07357 from the National Institute of llergy and Infectious Diseases. Dr. Martin's present address: Department of Microbiology, Faculty of Medicine, University of Panama, Panama, R.P. Requests for reprints should be sent to Dr. Schneidau. 875 identification of yeasts. The test described by Beijerinck 2 in 1889 is still being used, with modifications, by many investigators. In this technic, a pour plate of a heavy suspension of yeast cells is made, following which small amounts of various sugars are placed on the surface of the agar. Growth develops in the areas where the assimilable compounds were placed. jello and associates 1 recommended the use of wells in the agar of the petri dish, with subsequent addition of the carbon source, whereas Di- Menna 8 employed disks impregnated with the sugars, which were placed various distances apart on the agar surface. Bump and Kunz 5 added drops of the test carbohydrate to different sections on the surface of the agar. This paper describes an assimilation test for yeasts which is simple to perform, easily readable, and less subject to errors
876 MRTIN ND SCHNEIDU Vol. 53 of interpretation than some of the methods currently in use. Materials and Methods Fifty-seven strains of Candida, representing seven species frequently associated with clinical specimens of human origin, were studied. The sources of the strains are listed in Table 1. The assimilation test was performed in the following manner. Yeast-nitrogen base (basal medium) was prepared as a 6.7% solution in distilled water and sterilized by Seitz filtration. Dextrose, maltose, sucrose, lactose, galactose, cellobiose, raffinose, and trehalose at 20% concentrations were also sterilized by filtration and utilized as test carbon sources. gar at 2% concentration was completely dissolved by heating and dispensed in 10 ml. quantities to 20 mm. by 150 mm. test tubes, after which the tubes were autoclaved at 15 lb. for 15 min., then cooled to 50 C. To each test tube, 1.0 ml. basal medium and 0.5 ml. carbon source were added aseptically, thereby constituting a 1% sugar solution, approximately. The control tubes contained agar, basal medium, and 1.5 ml. distilled water. The medium was allowed to cool thoroughly in a slanting position and stored at 4 C. ll cultures were maintained on Sabouraud's dextrose agar medium and tested after being allowed to grow for five or six days. Clinical specimens were obtained in pure culture, maintained on Sabouraud's dextrose agar, and tested in the same manner. To prepare the test organisms, three colonies of yeast were suspended in 5 ml. sterile physiologic saline solution. One-half milliliter of this suspension was pipetted onto the surface of each of the agar slants containing the various sugar sources, as well as into a control tube. Care was taken to get the inoculum in contact with most of the surface of the agar slant. The slants were incubated at 25 C. and observed for growth at 48 hours, four to five days, and finally, ten days. ssimilation was considered positive when abundant growth appeared on the test medium with negligible or no growth in the control tube. ssimilation was considered negative when there was no significant difference between growth of the organisms on the test medium and growth in the control medium. ll strains were also tested for fermentation of dextrose, maltose, sucrose, and lactose, for chlamydospore formation on cornmeal agar, and for germ tube production in egg white medium, as described by Buckley and Van Uden. 4 The results of these tests were compared with the data obtained in the assimilation tests. Results The reactions of the strains of Candida studied appear in Table 2. It can be seen from these data that it is easy to distinguish C. albicans from C. stellatoidea by the sugar assimilation test, whereas they cannot always be distinguished on the basis of their activity in germ tube production, chlamydospore formation, or fermentation of sugars. Furthermore, it is also possible to differentiate C. krusei and C. parakrusei by their characteristic sugar assimilation patterns. They have similar fermentation reactions, which makes identification on that basis difficult. C. albicans and C. parakrusei had identical assimilation spectrums and required other tests, i.e., sugar fermentation, chlamydospore formation and germ tube production, for their identification. ll of the other species of Candida studied had characteristic assimilation patterns. Discussion The sugar fermentation, germ tube production, and chlamydospore formation do not provide exact, precise means of distinguishing between C. albicans and C. stellatoidea, inasmuch as the fermentation
June 1970 SSIMILTION TEST FOR CNDID IDENTIFICTION 877 Table 1. Sources of Candida Strains Studied C. albicans Species and Strain No. B612, B613 65-89, Mur DS378, DS374, DS385, 216, 377, V Hosp., Roth 20M22, H. llemand 6871, 6910 44, C Y326 C. krusei 331, B 7183 C. parakrusei TCC 10232, B 8556, 1044, 5536, 375, 2125, 508, 36, 41, 42, 6563, 6648, 6866, 6964, 7002, 7003, 7031, 7033, 7034, 7035, 7036, 7083, 7084 C. tropicalis B396 52,53 C. psetulolropicalis, B C. guilliermondii, B YM55 7005 Source * Dr. M. Sue Ivensf J * National Communicable Disease Center, tlanta, Georgia. f Louisiana State University School of Medicine, New Orleans, La. X Oak Ridge ssociated Universities, Oak Ridge, Tenn. patterns of the two organisms can be, and many times are, identical. For example, five strains of C. albicans had a fermentation pattern similar to that of the four strains of tested (Table 2). lso, although C. albicans usually produced abundant chlamydospores on cornmeal agar and only rarely produces these structures, there are strains of C. albicans that are poor chlamydospore producers. Two strains of formed occasional chlamydospores on corn-
Table 2. Results of Chlamydospore Formation, Germ Tube Production, and Sugar Fermentation and ssimilation Tests of Representative Strains of Candida* Organism Chlamydospore Germ Tube Formation Production Fermentation ssimilation Dex- Malt- Sutrose ose crose Lactose Dex- Malt- Su- Lac- Galac- Cello- Raffi- Trehalose ose crose tose tose biose nose lose C. albicans (9 f C. albicans (5 (strain 44) (strain C) (2 C. krusei (4 C. parakrusei (14 C. parakrusei (11 C. guilliermondii (4 C. tropicalis (4 C. pseudotropicalis (2 _ * = acid and gas; = acid only; = few produced or formed, t Strains giving identical reactions are grouped. _ - - - - - - - - - - - - - 00 00 H o n X w 3 z
June 1970 SSIMILTION TEST FOR CNDID IDENTIFICTION 879 meal agar, whereas three produced a rare germ tube on egg white incubated at 37 C. However, these two species can be distinguished readily by their differential behavior on sugar assimilation, since C. albicans assimilates sucrose, and does not. Similarly, C. krusei, C. parakrusei, and C. guilliermondii may have the same sugar fermentation patterns. The four strains of C. krusei analyzed were indistinguishable by sugar fermentation from 14 of the 25 strains of C. parakrusei tested (Table 2), whereas the carbon assimilation behavior of each of these species was characteristic and permitted precise classification. The four strains of C. guilliermondii studied fermented dextrose and sucrose, producing acid and gas, and therefore behaved differently from the strains of C. krusei and C. parakrusei tested. Nevertheless, C. guilliermondii is known to ferment only dextrose at times, 1 in which case this test would not suffice to separate it from either C. krusei or C. parakrusei. The strains of C. tropicalis studied could be identified by their ability to assimilate cellobiose, but some strains may fail to utilize this sugar. 1 However, all strains of C. tropicalis had a characteristic fermentation pattern so that, for this species, sugar fermentation does provide an adequate diagnostic tool. Similarly, C. pseudotropicalis had a unique fermentation pattern with its fermentation of lactose, a sugar neither fermented nor assimilated by any of the other species of Candida studied. C. albicans and C. parakrusei behave identically on sugar assimilation, and for these two species germ tube production, chlamydospore formation, and sugar fermentation are necessary for identification. The technic described has an advantage over some of the other methods in that each sugar is placed in a separate, individual tube, so that the diffusion of adjacent sugars into the surrounding medium is prevented. Such diffusion sometimes occurs when performing the test in petri dishes, be it in wells, disks, or just by dropping the sugar onto the surface of the agar. The use of a sugar-free control tube eliminates false-positive interpretations that may result when traces of nutrient are carried over in the inoculum. References 1. jello, L., Georg, L. K., Kaplan, W., and Kaufman, L.: Laboratory Manual for Medical Mycology. U. S. Dept. of Health, Education, and Welfare, C.D.C., tlanta, Georgia, pp. El- E25. 2. Beijerinck, M. W.: L'auxanographie, ou la methode d l'hydro diffusion dans la gelatine appliqu e aux recherches raicrobiologiques. rch. Nederl. Sci. 23: 367-S72, 1889. 3. Bonfante, R.: Development and evaluation of a rapid identification test for Candida albicans. Mycopathologia 34: 33-39, 1968. 4. Buckley, H. R., and Van Uden, N.: The identification of Candida albicans within two hours by the use of an egg white medium slide preparation. Sabouraudia 2: 205-208, 1963. 5. Bump, C. M., and Kunz, L. F.: Routine identification of yeasts with the aid of molybdateagar medium. ppl. Microbiol. 16: 1503-1506, 1968. 6. DiMenna, M. E.: search for pathogenic species of yeasts in New Zealand soils. J. Gen. Microbiol. 12: 54-62, 1955. 7. Lau, H. S.: Evaluation of media and methods for chlamydospore formation by Candida albicans. Techn. Bull. Regist. Med. Techn. 50: 132-134, 1968. 8. Mackenzie, D. W. R.: Serum tube identification of Candida albicans. J. Clin. Path. 15: 563-565, 1962. 9. Svobodova, Y and Chmcl, L.: method for the rapid identification of pathogenic yeastlike organisms. Mycopathologia 26: 403^109, 1965. 10. Taschdjian, C. L., Burchall, J. M., and Kozinn, P. J.: Rapid identification of Candida albicans by filamentation on serum and serum substitutes. mer. J. Dis. Child. 99: 102-105, 1960.