NUTRITIONAL REQUIREMENTS OF DERMATOPHYTES IN CONTINUOUS SHAKE CULTURE* F. RAUBITSCHEK, M.D.

NUTRITIONAL REQUIREMENTS OF DERMATOPHYTES IN CONTINUOUS SHAKE CULTURE* F. RAUBITSCHEK, M.D. Studies of the nutritional requirements of dermatophytes have been made by various workers using stationary cultures on solid and liquid media. Conclusions as to the ability of these fungi to grow on inorganic nitrogen in the presence of a suitable carbohydrate source are contradictory. Various sporulating strains of Trichophyton and Microsporon have been found to grow under these conditions. (Bocobo and Benham (3), George (4, 5), Giblett and Henry (6), Silva and Benham (19), and Bereston (2)). However, only pleomorphic strains of T. mentagrophytes grew on inorganic nitrogen, while granular sporulating strains did not according to others (Mosher et al. 1936 (13), Robbins and Ma (18), Nickerson (14), McVeigh and Campbell (11), and Stockdale (20)). Technical considerations such as criteria for evaluating growth, the use of media only partly defined chemically, and variations in the size of inocula may be responsible for some of these discrepancies. A method which yields a constant amount of growth from a given inoculum under fixed conditions is the continuous shake culture. In this method, first used for the culture of dermatophytes by Wyss et al. (22), the organisms grow in the form of well defined pellets whose size depends on the type of organism, on the composition of the medium and on the size of the inoculum. Microscopically those pellets consist of a maze of septate branching hyphae breaking up into arthrospores, and varying amounts of chlamydospores (Raubitschek (17)). Neither macro- nor micro-conidia nor other structures such as are found attached to the aerial growth on stationary culture occur in these pellets. Thus the type of growth resembles that of the parasitic life phase of the fungi, found in scales, hairs and nails, and will therefore be referred to as the "pseudoparasitic" phase. This investigation was initiated to study some of the nutritional requirements of strains of dermatophytes in this "pseudoparasitic" life phase, grown under continuous shaking conditions. Materials and Methods. Strains of dermatophytes freshly isolated from human lesions and cultured on Sabouraud's dextrose agar (Difco) were used. The strains were granular in type of growth and showed luxuriant sporulation. The inoculum was prepared by adding 5 ml of sterile saline to a slant, detaching part of the aerial growth with a straight wire into the saline, shaking, and then pouring into sterile centrifuge tubes. This procedure was performed as quickly as possible to minimize elution of nutrients from the agar. The resulting cell suspension was centrifuged at 3000 rpm for three hours, and the supernatant was discarded. (The prolonged centrifugation was necessary since after shorter * From the Department of Dermatology and Venereology (Dr. A. Dostrovsky, Director), Hebrew ljniversity-hadassah Medical School, Jerusalem, Israel. Received for publication January 6, 1955. 83

84 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY periods of centrifugation part of the fungal elements remained floating on the surface.) The sediment was washed twice with saline and the final cell residue suspended in two ml of sterile saline and kept in the refrigerator as stock suspension. The inoculum consisted of 0.2 ml of this suspension per 100 ml of medium. The dry weight of 0.2 ml of stock suspension varied from 1.2 to 2.4 mg. The use of smaller inocula resulted in great variation in the final total weight, while larger inocula yielded pellets which varied greatly in size. In order to obtain comparable results the entire suspension from a single slant was inoculated into as many different media as possible. Occasionally a stock suspension was insufficient for inoculation of all media. The inoculated flasks were shaken continuously on a Ross-Kershaw type shaking machine, 140 strokes per minute, at 30 32 C for 12 days. The contents of a flask were then quantitatively transferred to weighed filter paper, washed with distilled water, air dried, dried further at 50 C over CaCl2 in vacuo and weighed. Each experiment was done in triplicate, and several experiments were repeated. The growth from three flasks was calculated in terms of the average weight of dried mycelium per 100 ml of medium. Media. The basal medium contained per liter of triple distilled water: K2HPO4, 0.2%; MgSO47H2O, 0.005%; CaCl2, 0.005%. This basal salt solution with additional nutrients was dispensed into 250 ml Erlenmeyer flasks in 100 ml quantities and autoclaved. The compounds investigated were: ammonium sulfate, 0.3%; dextrose, 1.0%; soluble yeast extract (Difco), 0.01%; Bacto pepton (Difco), 1.0%; and a local brand of casein hydrolyzate (not vitamin free), 0.6%. All chemicals used were of analytical reagent quality. All glassware was washed in acid. Organisms. Three strains of Trichophyton mentagrophytes, two strains of Trichophyton rubrum, two strains of Trichophyton tonsurans, and one strain of Epidermophyton floccosum were used. RESULTS All strains of T. mentagrophytes, T. rubrum and T. tonsurans showed growth on inorganic nitrogen even in the absence of carbohydrate (Table I). Addition of carbohydrate to the media containing the inorganic nitrogen resulted in marked increase of growth. However, E. floccosum showed no growth in the presence of ammonium sulfate alone, although it did when carbohydrate was added. Yeast extract added to the ammonium sulfate-carbohydrate medium caused distinct additional growth of two of the three strains of T. mentagrophytes, one of the two strains of T. rubrum and of the E. floccosuni strain. The two T. tonsurans strains were unaffected by the yeast extract addition. Basal salt solution with the addition of casein digest or pepton were about as effective for growth as the ammonium sulfate-carbohydrate-yeast extract medium. Visible growth started within two to six days, depending upon the composition of the medium, and consisted of pellets 2 5 mm in diameter. In the pepton and in the casein digest media growth tended to begin early, showed a marked tendency to lyse, and the medium was turbid at the end of the shaking period

NUTRITIONAL REQUIREMENTS OF DERMATOPHYTES 85 TABLE I Growth yield of dermatophytes under continuous shaking conditions Basal Salt Solution with the Addition of Organism 0.3% Ammonium Ammonium If sulfate, 1.0% 0.6%?asein 1.0% Pepton sulfate i.0, dextiose dextrose, 0.oi% Y ro yza T. mentagrophytes 5073 19.5 263 19 100.6 144 165 172 14.5 91.6 188 172 217 7'. rubrum 5131 18 44 119 189 152 16 43 60 T. tonsurans 3945 17 72.6 79 15.3 59 62 E. fioccosum n.g. 33 111 110 Results are expressed in terms of mg of dried myceliurn per 100 ml of medium, average from three flasks (n.g. = no growth). Shaking was continuous for 12 days at 30 32 C. Inoculum consisted of 0.2 ml stock cell suspension per flask of 100 ml medium. although clear after filtration. In casein digest, in pepton, and in yeast extract media, there was additional fungal growth in a circular band around the flask. The site of this band corresponded to the highest level reached by the medium during shaking. Microscopically and macroscopically the growth in the band was similar in all respects to the growth obtained in stationary culture. On these media the growth was determined by weighing together both pellets and bands. Pellets grown previously in pilot experiments were transferred to Sabouraud's dextrose agar (Difco) slants and developed invariably into the sporulating granular form of growth typical of the respective organism. COMMENT The present investigation has demonstrated that some fungi are capable of proliferating in a basal medium containing only inorganic nitrogen. The carbon required for cell constituents was probably derived from the atmosphere. (Nitrogen fixation from the atmosphere by Sporotrichum species has been demonstrated by Lurie (10)). Energy for cell synthesis was most probably provided by the oxidation of ammonia, of atmospheric carbon compounds and also from high energy bond containing compounds within the cells of the inoculum. Experimental demonstration of these reactions is lacking at present but will be sought in further studies. This autotrophic behavior of the "pseudoparasitic" phase produced in shake culture indicates greater synthetic powers than are reported for the granular and the pleomorphic forms of the saprophytic life phase of dermatophytes. The greater growth on dextrose and ammonium sulfate compared with that on

86 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY ammonium sulfate alone suggests that carbohydrate is utilized by dermatophytes grown under shake conditions. Carbohydrate utilization by dermatophytes has been reported by others (Hopkins and Iwamato (7, 8), Tate (21), Lewis and Hopper (9), Nickerson and Chadwick (16), Nickerson (15), Melton (12), Barlow and Chattaway (1)). Growth of some strains of fungi under shake conditions was increased by the addition of yeast extract to the basal medium containing ammonium sulfate and dextrose. A similar large yield was also obtained in cultures grown on basal salt solution and casein digest or pepton. The quantity of nitrogen in the two latter media was equivalent to the amount of nitrogen present in the ammonium sulfate medium. These findings suggest that yeast extract, casein digest and pepton may contain a growth-promoting substance or substances. SUMMARY 1. Under continuous shaking conditions dermatophytes were grown in a form morphologically similar to that found in lesions of man and animal. 2. All strains of dermatophytes investigated, except B. fioccosum, were able to grow in a basal salt solution containing ammonium sulfate without carbohydrate. All strains grew well on inorganic nitrogen in the presence of dextrose. 3. Addition of yeast extract to the ammonium sulfate-dextrose medium increased the growth of some organisms. 4. Casein hydrolyzate or pepton added to basal salt solution were able to support growth of some fungi to the same extent as a yeast extraet-ammonium sulfate-dextrose medium. Thanks are due to Miss E. Sheinberg for technical help. REFERENCES 1. BARLOW, A. J. AND CRATTAWAY, F. W.: Effect of iohibitors on the metabolism of microsporon. Biochem. J., 5: XXXI, 1953. 2. BERR5T0N, E. S.: The vitamin, amioo acid and growth requirements of the genus microsporum. J. Invest. Dermat., 20: 461 469, 1953. 3. Bocoo, F. C. AND BENHAM, R. W.: Pigment production in the differentiation of tnehophyton mentagrophytes and trichophyton rubrum. Mycologia, 41: 291 302, 1949. 4. GEOEG, L. K.: Influence of nutrition on growth and morphology of dermatophytes. Tr. New York Acad. Sc., Ser. II, 11: 281 286, 1949. 5. GEOEG, L. K.: The relation of nutrition to the growth and morphology of triehophyton violaceum. I. The vitamin and amino acid requirements of T. violaeeum. Myeologia, 43: 297 309, 1951. 6. GIBLETT, E. R. AND HENRY, B. S.: Physiological studies on the genus microsporum. J. Invest. Dermat., 14: 377 386, 1950. 7. Hovair'is, J. C. AND IwAluATo, K.: Fermentation reactions of the ringworm fungi. I. Arch. Dermat. & Syph., 8: 619 624, 1923. 8. HOPKINS, J. C. ANn IwAMATO, K.: Fermentation reactions of the ringworm fungi. II. Arch. Dermat. & Syph., 8: 838 843, 1923. 9. LEWIS, C. M. AND HOPPER, M. E.: Pigment production by fungi. Arch. Dermat. & Syph., 44: 453 460, 1941. 10. Luarz, H. J.: Sporotriehon species: their nitrogen metabolism. Myeologia, 43: 117, 1951.

NUTRITIONAL REQUIREMENTS OF DERMATOPHYTES 87 11. McVaiou, I. AND CAMPBELL, F.: The growth of trichophyton mentagrophytes and five of its variants as affected by several nitrogen sources. Mycologia, 42: 451 469, 1950. 12. MELTON, F. M.: The effect of various substances on the oxygen uptake of microsporum canis grown in submerged culture. J. Invest. IDermat., 17: 27 34, 1951. 13. MOSHEE, W. A., SAUNDERs, D. H., KINOEEY, L. B. AND WILLIAMS, R. J.: Nutritional requirements of the pathogenic mold, trichophyton interdigitale. Plant Physiol., 9: 795 806, 1936. 14. NIcKEE50N, W. J.: Pathogenic fungi. Chronica Botanica Comp., Waltham, Mass., 1947. 15. Nzcicrtsos, W. J.: Mutation and gains in synthetic abilities among fungi pathogenic for man. Rep. Proc. Fourth International Congress for Microbiology, Copenhagen, 382 384, 1947. 16. NIcKER50N, W. J. AND CHADWIcK, J. B.: On the respiration of dermatophytes. Arch. Biochem., 10: 81 100, 1946. 17. RAUBIT5cIIEK, F.: Unpublished observations, 1953. 18. ROBBINS, W. J. AND MA, R.: Growth factors for trichophyton mentagrophytes. Amer. J. Bot., 32: 509 523, 1945. 19. SILvA, M. AND BENHAM, R. W.: Nutritional studies of the dermatophytes with special reference to Trichophyton Megnini Blanchard 1896 and Trichophyton Gallinac (Megnin 1881) Comb. nov. J. Invest. Dermat., 18: 453 472, 1952. 20. STOcKDALE, P. M.: Requirements for the growth and sporulation of trichophyton persicolor. J. Gen. Microbiol., 8: 434 441, 1953. 21. TATE, P.: On the enzymes of certain dermatophytes. Parasitology, 21: 31 54, 1929. 22. WTss, 0., LUDWIG, B. J. AND JOINEE, H. H.: The fungistatic and fungicidal action of fatty acids and related compounds. Arch. Biochem., 7: 415 425, 1945.