ROUTINE NUTRITIONAL TESTS FOR THE IDENTIFICATIONOF DERMATOPHYTES

LUCILLE K. GEORG AND LAVERNE B. CAMPCommunicable Disease Center, Public Health Service, U. S. Department of Health, Education, and Welfare,

Atlanta, Georgia

Received for publication November 16, 1956

Dermatophytes are usually identified on thebasis of the gross and microscopic cultural char-acteristics that develop on Sabouraud's dextroseagar. Most isolates develop colony forms, pig-mentation, and spores which are diagnostic forthe species. However, many atypical strains areencountered. In addition, there are a number ofspecies which seldom produce spores and whosecolonies resemble each other so closely that theycan not be identified solely on the basis of mor-phological criteria. As an aid in the identificationof the dermatophytes, particularly variousTrichophyton species, a number of practical physi-ological tests have been developed.

MATERIALS AND METHODS

The methods used are based essentially on theones developed during the study of the nutri-tional requirements of various dermatophytespecies (Georg, 1950, 1951, 1952; Swartz andGeorg, 1955).

The basal media. The two basal media used inthe tests, casein and NH4NO3 agar, were madewith (1) a "washed agar" that had been preparedin the laboratory by washing lots of agar (Difco)15 times in distilled water, immersion in boiling95 per cent ethyl alcohol, and drying, (2) threedifferent samples of "purified agar" prepared byDifco Laboratories, and (3) untreated agar(Difco).Experiments were also carried out to deter-

mine the effect of trace elements in the growth ofthe fungi. The two basal media were preparedwith and without the addition of a trace mineralsolution (Georg, 1952). The only salts added tothe media were MgSO4 and KH2PO4.The media prepared with the different nitrogen

sources and with and without the trace mineralsolution were used in nutritional tests with severaltypical strains of Trichophyton verrucosum, Tri-chophyton tonsurans, and Tricophyton megninii.These species produce characteristic growth pat-terns in the presence of certain vitamins and amino

acids. The media with the variously preparedagars were assayed for traces of thiamin by usingspores of Phycomyces blakesleeanus.

Vitamins and amino acids. Tests were per-formed using vitamin and amino acid solutionsprepared in acidified water and sterilized byfiltration through Seitz filters. Parallel tests wererun using similar solutions sterilized by auto-claving at 120 C for 10 min.

RESULTS

It was found that various types of "purifiedagars" contained thiamin, although in quantitiesless than in the agar routinely used in mycologicalor bacteriological studies. However the testmedia prepared with agar (Difco) were satis-factory for use in the nutritional tests. It wasfound that the addition of trace mineral elementsto the media had no visible effect on the growthof the dermatophytes. Autoclaving of the vita-min and amino acid solutions did not diminishtheir activity. On the other hand, it was foundessential to acid clean glassware in order to re-move traces of thiamin which would vitiate thetests.

Procedure for routine nutritional tests. Stockmedia consisted of (1) casein agar, and (2) am-monium nitrate agar.

Casein agar.

Casein, 10 per cent acid hydrolyzed,vitamin free (Nutritional Biochemi-cals Co.) or Casamino Acid (Difco),vitamin free 2.5 g ............. 25.0 ml

Glucose... .......... 40.0 gMgSO4..............0. 1 gKH2PO4 .............. 1.8 gAgar (Difco) ...... ....... 20.0 gWater (distilled) ............. q.s. 1,000.0 ml

Dissolve by heating and distribute in 100 mlquantities into flasks and autoclave at 120 C for15 min. The contents of several of the flasks may

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be distributed into test tubes and slanted for useas vitamin free controls. Other flasks may bekept for preparing the different media containingvitamins as needed.Ammonium nitrate agar. The preparation of

this medium is similar to that of the casein agarexcept that 1.5 g NH4NO3 is substituted for thecasein.

Test media. The test media are prepared byadding vitamins to the casein medium and his-tidine to the NH4NO3 agar to give the media thefollowing final concentrations: (1) Thiamin-casein agar, thiamin hydrochloride 0.2 ,ug/ml,(2) Inositol-casein agar, i-inositol 50 ,ug/ml, (3)Thiamin-inositol casein agar, thiamin 0.2 ,ug/mland inositol 50 ,ug/ml, and (4) Histidine-am-monium nitrate agar, L-histidine 30 ,ug/ml.The growth factors are added to melted stock

media. These are then distributed into tubes,autoclaved at 120 C for 10 min, slanted andstored at 5 C for use as needed.

Inoculation of nutrition tubes. The inoculummay be taken from the growth obtained on anyof the usual isolation media. Care must be taken,however, to take only a small fragment of thegrowth and thus avoid carrying any significantquantity of the medium to the nutrition tube.

Nutritional patterns for the dermatophytes. Thenutritional patterns were found to fall into 4groups.Group I. The Trichophyton species in this

group are those which seldom produce spores ordistinctive pigments, and whose colonies resembleone another so closely that they can not beidentified by morphological criteria. These speciesare: (a) T. verrucosum, (b) T. schoenleinii, and(c) T. concentricum. They are usually identifiedon the basis of clinical source as well as geo-graphical origin.

(a) T. verrucosum:-Nutritional studies of T.verrucosum have been reported previously (Georg,1950). Since this publication, 100 strains havebeen identified and studied by this laboratory.Seventy-four had been isolated from humans, 23from cattle, 2 from donkeys, and 1 from a horse.None of the strains grew on either NH4NO3 or

casein vitamin free agar media. Eighty-fourstrains were shown to require thiamin andinositol for growth. Sixteen strains required onlythiamin. Additions of other water soluble vita-

TABLE 1Growth patterns of Trichophyton verrucosum, Tri-chophyton schoenleinii, and Trichophyton con-centricum. (This is a four tube test incubated at37 C and read after 7 and 14 days)

Dermatophyte

Trichophyton ver-rucosum (100strains studied)84%.........16%.........

Trichophytonschoenleinii (50strains studied)2 exceptions-see text.......

Trichophyton con-centricum (19strains studied)50%.........50%.........

Test Media

Casein

00

4+

4+2+

* i Indicates a traceabout the inoculum.

Casein +inositol

4-*0

4+

4+2+

Casein +thiamine

04+

4+

4+4+

Casein +thiaminand

inositol

4+t4+

4+

4+4+

of submerged growth

t 4+ Indicates maximum growth for that seriesof tubes, growth in other tubes being judged bycomparison.

mins,' singly or in combination with the requiredvitamins, produced no effect on the growth.

Further tests showed that molecular thiaminwas required by all strains studied. Thiamincould not be substituted by either pyrimidine orthiazole compounds or a combination of these.These findings are similar to those obtained byother workers (Robbins et al., 1942, Burkholderand Moyer, 1943; Schopfer and Blumer, 1943;Mackinnon and Artagaveytia-Allende, 1948). Thestrain studied by Robbins et al. was shown to re-quire 3 vitamins for growth: thiamin, inositol,and pyridoxine. Biotin appeared to be an addi-tional factor for a strain studied by Schopfer andBlumer. Such requirements were not foundamong the 100 strains included in this study.The diagnostic nutritional patterns of T. ver-

I Calcium pantothenate 100 ,ug/ml, riboflavin100 Ag/ml, p-aminobenzoic acid 100 ug/ml, nico-tinic acid or nicotinamide 100 ,g/ml, biotin 0.05,ug/ml, choline chloride 100 jAg/ml, and folic acid100 /Ag/ml.

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.A

c D

Figure 1. Four tube nutritional test: Left to right, (1) casein, (2) casein + inositol, (3) casein + thi-amin, (4) casein + thiamin and inositol. A. Trichophyton verrucosum strains which require both thiaminand inositol. B. Trichophyton verrucosum strains which require thiamin only. C. Trichophyton con-centricum strains which are autotrophic for the vitamins. D. Trichophyton concentricum strains whichare stimulated by thiamin. E. Trichophyton schoenleinii strains which are autotrophic for the vitamins.

rucosum may be demonstrated by a four tubetest as indicated in table 1.

(b) T. schoenleinii:-Preliminary studies of thenutritional requirements of T. schoenleinii, uti-lizing 6 strains, have been reported previously(Georg, 1950). Since then, 50 additional strainshave been studied. Thirty-one were recent iso-lates from typical favic-type hairs, and 19 wereobtained from culture collections. Of these 50

strains, 48 or 96 per cent presented a similar nu-tritional pattern. Characteristically T. schoen-leinii was found to be autotrophic for the vita-mins and developed on both NH4NO3 and caseinvitamin-free agar media. Although several of theisolates grew very slowly and poorly on the vita-min-free media, definite growth could be easilyobserved. Additions of water soluble vitamins,singly and in combinations, had no effect on the

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growth of any of the 48 strains. Similar resultshave been reported by Drouhet and Mariat(1952).

T. schoenleinii can be readily differentiatedfrom T. verrucosum by the four-tube test as illus-trated in table 1. Since T. schoenleinii is auto-trophic for the vitamins, equal amounts ofgrowth occur on all 4 tubes.Two strains of T. schoenleinii were encountered

which presented different nutritional patterns.One strain, isolated from a typical case of favus,was exceptional in that its growth was stimulatedby the addition of thiamin. A second strain, iso-lated from a chronic case of favus, was unable togrow on the vitamin free media. Additions of thewater soluble vitamins did not allow the growthof this fungus, however, addition of 10 per centyeast extract (0.05 ml/100 ml) permitted itsgrowth on the NH4NO3 or casein media.

(e) T. concentricum:-Eighteen strains of T.concentricum were studied. Seventeen were ob-tained from culture collections. One had been re-

cently isolated from clinical material from a Mexi-can case of tinea imbricata. Of the strains fromculture collections, 7 originated from tineaimbricata infections in Central and South Amer-ica, 8 were from the South Pacific area. The originof 3 of the strains was unknown.

All of the strains were able to grow on eitherNH4NO3 or casein vitamin free media, however,the growth on the casein medium was consid-erably greater than that on the inorganic nitro-gen medium. Seven of the isolates were not stim-ulated by additions of the water soluble vitamins.Ten strains were definitely stimulated by thiamin.This stimulation was shown to be due to the thia-zole portion of the thiamin molecule. Equimolaramounts of thiazole, 4 methyl 5 b-hydroxyethylthiazole (Merck and Co.), produced the same

effect as the intact thiamin molecule. The variouspyrimidines tested were inactive. A similar re-

quirement for thiazole has been reported byDrouhet and Mariat (1952) for a strain of T.concentricum which they studied.

There was no correlation between the geo-

graphic source of the isolates and their nutri-tional requirements. The two nutritional patternsof T. concentricum as shown by a four-tube testare indicated in table 1.Group II. In this group are included: (a)

Trichophyton tonsurans, (b) T. mentagrophytes,and (c) T. rubrum. These are Trichophyton spe-

cies which usually produce microconidia, butonly occasionally produce macroconidia. Theircolonial forms and pigments are so variable thatthese fungi are frequently difficult to differen-tiate. A two-tube test with casein vitamin freeagar and a similar medium with added thiaminis useful in identifying these species.

(a) T. tonsurans:-Nutritional studies withthis fungus have been previously reported bySwartz and Georg (1955). To date 60 strains ofT. tonsurans isolated from hairs parasitized in anendothrix manner have been studied. All strainshave been shown to be autotrophic for the watersoluble vitamins with the exception of thiaminfor which they showed a partial requirement.Two or three times as much growth occurred oncasein agar with added thiamin as on controlcasein tubes. Equimolar amounts of severalpyrimidine compounds, such as 2 methyl-4-amino, 5-aminoethyl pyrimidine (NutritionalBiochemicals Co.) produced the same effect asthiamin. The various thiazole compounds testedwere inactive. Seven of the strains studied wouldnot grow on NH4NO3 medium even in thepresence of thiamin. These strains were shown tohave a requirement for amino acids of theornithine, citrulline, and arginine cycle. Findingssimilar to these have been reported by Burk-holder and Moyer (1943), Drouhet (1952), Drou-het and Mariat (1952), and Sullivan et al. (1954).

TABLE 2Growth patterns of Trichophyton tonsurans, Tri-chophyton mentagrophytes, and Trichophyton ru-brum. (This is a two tube test carried out atroom temperature and read at 7 to 10 days)

Test Media

Dermatophyte CaseinCasein +

thiamin

Trichophyton tonsurans (70strains studied) ...........:i to 1+ 4+

Trichophyton mentagrophytes(50 strains studied) ....... 4+ 4+

Trichophyton rubrum (50strains studied) ........ 4+ 4+

T. mentagrophytes may be differentiated fromT. rubrum by in vitro hair cultures (Ajello andGeorg, 1957). T. mentagrophytes forms perforatingorgans in human hair samples suspended in waterwithin 2 to 3 weeks. T. rubrum does not perforatehuman hair.

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A

Figure 2. Two tube nutritional test: Tube at left of each pair, casein; tube at right of each pair,casein + thiamin. A. Trichophyton rubrum: granular strain (pair at left), fluffy strain (pair atright). Both autotrophic for thiamin. B. Trichophyton mentagrophytes: granular strain (pair atleft), downy strain (pair at right). Both autotrophic for thiamin. C. Trichophyton tonsurans:Two strains. Both show stimulation by thiamin.

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The nutritional pattern of T. tonsurans as shownby a two-tube test is indicated in table 2.

(b) T. mnentagrophytes:-Sixty-nine strains ofT. mentagrophytes were included in this study. Ofthese, 38 strains weie derived from human cases

of ringworm and 31 strains from spontaneousringworm of animals which included: 6 dogs, 11wild mice, 7 wild iats, 3 chinchillas, 2 guinea pigs,1 kangaroo, and 1 horse. The amount of growthon NH4NO3 agar was small except in the case ofpartially pleomorphic strains which grew well on

this medium. All strains, however, developedrapidly on the casein vitamin free medium. Noneof the strains were stimulated by the additionsof the water soluble vitamins. These results are

similar to those reported by Robbins and Ma(1945), and M\IcVeigh and Campbell (1950),Drouhet and Mlariat (1952), and Silva and Ben-ham (1954). The lack of stimulation by thiaminis in contrast to that observed with T. tonsuransand is illustrated in table 2.

(c) T. rubrum:-Forty-four morphologicallytypical strains isolated from human ringwormNere studied. All of these strains were able togrow on vitamin free media. However, consider-ably greater growth occurred on the casein thanon the NH4NO3. No stimulation by any of thewater soluble vitamins was found. This corre-

sponds to the findings of Drouhet and 1\lariat(1952) and of Silva, Keston, and Benham (1955).The lack of stimulation by thiamin is in contrastto that observed in T. tonsurans. The nutritionalpattern as shown by the two-tube test is illus-trated in table 2.Thus T. rubrum and T. nmentagrophytes can not

be differentiated fromn each other by thesenutritional tests. Another type of physiologicaltest can be used, however, for their differentia-tion. This test is based on the fact that T.mentagrophytes perforates hairs in in vitro haircultures while T. rubrum does not. Details of thistest have been described bY Ajello and Georg(1957).Group III. Trichophyton species which seldom

produce microconidia but which usually developcharacteristically pigmented colonies are placedin this group. Occasionally, however, atypicalstrains are encountered and physiological testsmay be useful in distinguishing them from other(leimatophyte species. The dermatophytes in thisgr'oup are: (a) Trichophyton violaceumn, and (b)Trichophyton ferruigineuam2.

TABLE 3Growth patterns of Trichophyton ferrugineurn and

Trichophyton i4olaceurn

Test MediaDermatophyte

Casein Casein +thiamin

Trichophyton ferrugineumn (14strains sttudied ........... 4+ 4+

Trichophyton violaceum (13strains studied) ........... +i 4+

Although T. violaceum has a similar nutritionalpattern to that of T. tonsurans, it is not likely tobe confused with this organism. T. violaceumgrows extremely slowly, even in the presence ofthiamin, and usually prodtuces a small glabrouscolony with no spores. T. tonsurans grows rapidlyin the presence of thiamin and shows many micro-conidia in the surface growth.

(a) T. violaceum:-Nutritional studies on thisfungus have been previously reported by Georg(1951). Thirteen strains, 9 from culture collec-tions and 4 recent isolates from endothrixparasitized hairs, have been included in this study.All strains grew very poorly on both vitamin freemedia. All strains, however, were greatly stimu-lated by thiamin. This was particularly apparenton the thiamin enriched casein medium. Equalamounts of pyrimidine, such as 2-methyl-4-amino, 5-aminoethyl pyrimidine hydrochloiride,produced the same effect as thiamin. Variousthiazole compounds tested were inactive, aswere the other watei soluble vitamins. Thesefindings correspond to those of Drouhet andAMariat (1952). They are indicated in table 3.

(b) T. ferrugineum:-Fourteen morphologi-cally typical strains obtained from culture collec-tions were studied. All strains except one wereable to grow on the NH4NO3 vitamin free agar.The exceptional isolate was found to requir eL-leucine for growth. All strains grew well oncasein vitamin free media and were not stimu-lated by additions of any of the water solublevitamins as described above. The nutritionalpatterns of T. violaceum and T. ferrugineum aIeillustrated in table 3.Group IV. This group includes those

Trichophyton species which can be identifiedsolely on the basis of specific nutritional require-ments which serve to distinguish them from allother dermatophyte species. These species are:(a) T. megninii and (b) T. equin'irn.

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(a) T. megninii:-Nutritional studies on thisfungus have been previously reported by Georg(1952). Thirteen strains, 9 from culture collec-tions and 4 recently isolated strains from Israeland Sardinia, were studied. None of the strainsgrew on the NH4NO3 vitamin free medium,however, all,grew well on the casein vitamin freemedium. Further studies indicated that thisorganism requires L-histidine for growth. Thisnutritional pattern is illustrated in table 4. Noother dermatophyte species shows this regularrequirement for histidine.In the past T. megninii has been confused with

T. gallinae. However, T. gallinae possesses bothmorphological as well as physiological differenceswhich serve to differentiate it.

Nutritional studies on T. gallinae have beenreported previously by Georg (1952). Sevenstrains, 6 isolated from ringworm of chickens and1 strain from a human infection described byTorres and Georg (1956), have been studied. All ofthe strains grew well on both NH4NO3 and caseinvitamin free agar. None of the strains werestimulated by additions of amino acids or thewater soluble vitamins. These findings are similarto those reported by Silva and Benham (1952),and by Drouhet (1953). The nutritional pattern ofT. gallinae is included in table 4 for comparisonwith T. megninii.

(b) T. equinum:-Nutritional studies on thisfungus have been reported previously by Georg(1949) and Georg et al. (1957). Thirteen strains ofT. equinum were observed. All strains except onehave been isolated from cases of equine ringworm.The lone exception had been isolated from a dog.

TABLE 4Growth patterns of Trichophyton megninii and

Trichophyton gallinae

Test MediaDermatophyte NH4NOa

NH4NO3 + histidine

Trichophyton megninii* (13strains studied) .. 0 4+

Trichophyton gallinaet (7strains studied) ........ 4+ 4+

* No other dermatophyte shows this regular re-quirement for histidine.

t T. gallinae is included on this chart, as thisspecies has been confused with T. megninii in thepast.

.B.

C

Figure S. Two tube nutritional test: A. Tricho-phyton megninii. Tube at left NH4NO3, tube atright NH4NO3 + histidine. Shows complete re-quirement for histidine. B. Trichophyton equinum.Tube at left casein, tube at right casein + nico-tinic acid. Shows complete requirement for nico-tinic acid. C. Trichophyton violaceum. Tube atleft casein, tube at right casein + thiamin. Showsstimulation by thiamin.

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TABLE 5Growth pattern of Trichophyton equinum*

Test Media

Dermatophyte Casein +Casein nicotinic

acid

Trichophyton equinum (13strains studied) ........... 0 4+

Trichophyton mentagrophytest(50 strains studied) .... 4+ 4+

* No other dermatophyte shows this require-ment for nicotinic acid.

t T. mentagrophytes has been included on thischart, as this species has been confused withT. equinum.

None of the strains studied could grow onNH4NO3 or casein vitamin free media. The addi-tion of various vitamins, singly or in combinationas indicated above, showed that T. equinum has acomplete requirement for nicotinic acid. Thiscompound may be replaced by nicotinic amide orthe amino acid, L-tryptophan. The requirementfor nicotinic acid appears to be distinctive for thisparticular dermatophyte species. It may bedemonstrated by a two-tube test as indicated intable 5. T. mentagrophytes has been included inthis table since this fungus has been confusedwith T. equinum in the past.Another physiological test for the identifica-

tion of T. equinum is based on the fact that thisfungus, in contrast to T. mentagrophytes, will notgrow on autoclaved human hair suspended insterile water. It will, however, grow on auto-claved horse hair, or the hairs of other equines(Georg et al., 1957).

DISCUSSION

Simplification of the procedures for studyingdermatophytes from a nutritional point of view,allows the use of such tests for the routine identi-fication of dermatophyte species. Certain of thedermatophytes have very distinctive nutritionalrequirements which set them apart from all otherdermatophytes. For example, T. verrucosum has acomplete requirement for thiamin, or thiaminand inositol; T. equinum requires nicotinic acid;and T. megninii requires L-histidine for growth.One species, T. tonsurans, does not have completerequirements for any vitamin, but its growth isgreatly stimulated by additions of thiamin. Other

species such as T. mentagrophytes and T. rubrumappear to be completely autotrophic for thevitamins.

In the process of studying groups of strainsfrom culture collections by these nutritionaltests, several errors in classification have beenuncovered. A culture listed in the American TypeCulture Collection as T. equinum (ATCC 9870)was found to be completely deficient for thiaminand inositol and as a result was identified as T.verrucosum. In another case, a culture from theLondon School of Hygiene and Tropical Medicinelisted as T. concentricum (L. S. H. T. M., D793)was found to require both thiamin and inositol forgrowth and thus was identified as T. verrucosum.This finding was of especial significance becausethe culture had not been isolated from a case oftinea imbricata. It had been recovered in SouthAfrica from a meat inspector, who had had a deepfolliculitis of six weeks' duration on the cheek andintra-mandibular region. The geographic loca-tion of this case, as well as its clinical picture,gives weight to the re-identification of thisfungus as T. verrucosum. There is one publishedreport of the isolation of T. concentricum fromSouth Africa (Pijper, 1918), but the descriptionof the fungus isolated does not conform in anyway to the characteristics of this species. Thusthere is no proof that T. concentricum occurs inAfrica.

It was interesting to find that the T.concentricum strains from South America and theSouth Pacific had similar nutritional require-ments. This gives further weight to the beliefthat the causative organisms of "tokelau" in theSouth Pacific and of "chimb8re" in SouthAmerica are identical as suggested by Fonseca(1930) and Figueroa and Conant (1940).

SUMMARY

Practical nutritional tests have been describedas an aid in the identification of certaindermatophytes.Although the tests require the use of acid

cleaned glassware, the preparation of the mediaand the procedures for their use are quite simple.

These procedures are particularly valuable inthe identification of those dermatophytes whichseldom produce spores and whose colonies re-semble each other so closely that they can not beidentified on morphological criteria alone. It isalso an aid in the identification of morphologicallyatypical strains.

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Diagnostic nutritional patterns for the fol-lowing dermatophytes are presented: Trichophy-ton concentricum, Trichophyton equinum, Tri-chophyton ferrugineum, Trichophyton gallinae,Trichophyton megninii, Trichophyton mentag-rophytes, Trichophyton rubrum, Trichophytonschoenleinii, Trichophyton tonsurans, Trichophytonverrucosum, and Trichophyton violaceum.

REFERENCESAJELLO, L. AND GEORG, L. K. 1957 In vitro hair

cultures for differentiating between atypicalisolates of Trichophyton mentagrophytes andTrichophyton rubrum. Mycopathol. et My-col. Appl., 7, 3-17

BURKHOLDER, P. R. AND MOYER, D. 1943 Vita-min deficiencies of fifty yeasts and molds.Bull. Torrey Botan. Club, 70, 372-377.

DROUHET, E. 1952 Recherches sur la nutritiondes dermatophytes. II. Action des acidesamines sur la croissance et la morphog6nese.Ann. inst. Pasteur, 82, 348-355.

DROUHET, E. 1953 Recherches sur la nutritiondes dermatophytes. III. L'histidine, facteurde croissance des Trichophyton du groupe ro-saceum. Ann. inst. Pasteur, 85, 791-799.

DROUHET, E. AND MARIAT, F. 1952 Recherchessur la nutrition des dermatophytes. I. Rtudedes besoins vitaminiques. Ann. inst. Pas-teur, 82, 337-347.

FIGUEROA, H. AND CONANT, N. F. 1940 Thefirst case of tinea imbricata caused by Tri-chophyton concentricum Blanchard 1896, re-ported from Guatemala. Am. J. Trop. Med.,20, 287-303.

FONSECA, 0. 1930 Affinidades parasitologicas eclinicas entre o tokelau da Asia e da Oceaniae o chimber8 dos indigenas de Matto-Grosso.Rev. Medico Cir. Brasil, 38, 281-311.

GEORG, L. K. 1949 Conversion of tryptophaneto nicotinic acid by Trichophyton equinum.Proc. Soc. Exptl. Biol. Med., 72, 653-655.

GEORG, L. K. 1950 The relation of nutritionto the growth and morphology of Trichophy-ton faviforme. Mycologia, 42, 683-692.

GEORG, L. K. 1951 The relation of nutritionto the growth and morphology of Trichophy-ton violaceum. Part I. The vitamin andamino acid requirements of T. violaceum.Mycologia, 43, 297-309.

GEORG, L. K. 1952 Cultural and nutritionalstudies of Trichophyton gallinae and Tri-chophyton megnini. Mycologia, 44, 470-492.

GEORG, L. K., KAPLAN, W., AND CAMP, L. B.1957 Equine ringworm with special referenceto Trichophyton equinum. Am. J. Vet. Re-search (accepted for publication).

MACKINNON, J. E. AND ARTAGAVEYTIA-ALLENDE,R. C. 1948 Vitamin deficiencies of sevenstrains of ectothrix, large-spored Trichophy-tons isolated from man and cattle. J. Bac-teriol., 56, 91-96.

MCVEIGH, I. AND CAMPBELL, F. 1950 Thegrowth of Trichophyton mentagrophytes andfive of its variants as affected by severalnitrogen sources. Mycologia, 42, 451-469.

PIJPER, A. 1918 Tinea imbricata in SouthAfrica. J. Trop. Med. Hyg., 21, 45-47.

ROBBINS, W. J. AND MA, R. 1945 Growth fac-tors for Trichophyton mentagrophytes. Am.J. Botany, 32, 509-523.

ROBBINS, W. J., MACKINNON, J. E., AND MA, R.1942 Vitamin deficiencies of Trichophytondiscoides. Bull. Torrey Botan. Club, 69,509-521.

SCHOPFER, W. J. AND BLUMER, S. 1943 Zur Wirk-stoffphysiologie von Trichophyton albumSab. Ber. schweiz. botan. Ges., 53, 409-465.

SILVA, M. AND BENHAM, R. W. 1952 Nutritionalstudies of the dermatophytes with specialreference to Trichophyton megnini Blanchard1896 Trichophyton gallinae (Megnin 1881)Comb. nov. J. Invest. Dermatol., 18, 453-472.

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