Sabouraudia (1976), 14, 287-297 A N A U T O M A T E D R A D I O M E T R I C M I C R O A S S A Y OF F U N G A L G R O W T H : Q U A N T I T A T I O N O F G R O W T H OF 1". M E N T A G R O P H Y T E S S. J.
QUALMAN,H.
E. JONES and W. M. ARTIS
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*Department of Dermatology, Immunodermatology Laboratory, Kresge H R4010 University of Michigan Medical Center, Ann Arbor, Mich. 48109 An automated radiometric microassay of the growth of Trichophytonmentagrophytesand other filamentous fungi is described. The assay is based upon the incorporation of 14C(U) glucose into the organism. Fractionation studies indicate that 73% of the label is found in trichloroacetic acid-insoluble macromolecular components of the mycelium. Incorporation of label directly correlated with growth as estimated by visual scoring of turbidity and as recorded in photomicrographs. Incorporation of 14C(U) glucose delineated a lag, exponential and stationary or plateau phase of growth. These phases could be completely inhibited by the antifungal agent toluaftate. It was concluded that the growth of filamentous fungi can be successfully monitored by the radiometric method described. Moreover, this method is sensitive, accurate, reproducible, rapid and free of the variability inherent in many traditional estimates of growth. For unicellular organisms like bacteria, an increase in population size is an accepted functional definition of growth (Stanier, D o u d o r o f f & Adelberg, 1963). Changes in population size can be measured b y direct counting and m a y be estimated b y culture turbidity or dilution plating techniques. G r o w t h of fungi, because it occurs b y apical h y p h a l extension in filamentous forms and b u d d i n g in the yeast forms, is not definable by a n increase in population size. I t is limited to a measurement of mass. Direct m e a s u r e m e n t o f mass (e.g., d r y weight deternaination) is an accurate b u t a w k w a r d a n d troublesome procedure. Most conventional measurements of fungal growth, therefore, are based u p o n indirect estimates of mass such as colony size or culture turbidity. These estimates are frequently visual and, as such, inherently subjective since growth m a y v a r y with the eye and bias of the observer. These techniques also necessitate prolonged culture of the organism due to their dependence on visible growth. (Benitez, M e d o f f & K o b a yashi, 1974). Reports have appeared in the literature in which the in vitro growth of fungi was monitored via changes in the q u a n t i t y of a structural c o m p o n e n t or in the binding of a radio-labeled precursor. Benitez et al., (1974) and Kobayashi, Medoff, Schlessinger, K w a n & Musser (1972) used the incorporation of radioactive amino acids into proteins, a n d radioactive nucleotides into ribonucleic acid as a measurement of viability of the yeast forms ofHistoplasma capsulatum and Blastomyces dermatitidis which might imply a measu r e m e n t of growth. Swanson & Stock (I966) observed that both the total c a r b o h y d r a t e and the total protein content of T. mentagrophytes var. quinckeanum closely paralleled changes in its mycelial weight. Noguchi, Banno, W a t a n a b e , N o z a w a & Ito (1975) extended this observation by extracting and quantitating the sugar c o m p o n e n t of T. mentagro*Address all correspondence and reprint requests to H. E. Jones, M.D., Division of Dermatology, Emory School of Medicine, Room 215, Woodruff Memorial Building, Atlanta B.A. 30322. 287
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phytes at different times during its growth phase. They found that the content of neutral sugar in the mycelium was directly proportional to mass. Olofinboba (1973) utilized incorporation of 14C(U) glucose by Botyrodiploidia theobromae as a means of following quantitative changes in the carbohydrate content of the fungus during its growth cycle. Changes in the amount of bound glucose were found to reflect changes in the weight of dried mycelium. Because this laboratory is concerned with factors which can influence the growth of dermatophytic fungi, we felt the necessity of developing a sensitive technique to accurately monitor fungal growth. The above observations suggested to us that growth of the pathogenic fungus T. mentagrophytes could be followed by an assay based on the assumption that a radio-labeled sugar would be incorporated into mycelial structural macromolecules. This paper describes an automated microassay of the growth of 7". mentagrophytes var. granulare. It is based upon the incorporation of 14C(U) glucose into trichloroacetic acid (TCA)-insoluble mycelial material. To our knowledge, this is the first report of any attempt to measure dermatophyte growth using radiometric techniques. The utility of this technique, however, is not limited to quantitation of growth of T. mentagrophytes. We also have found that this automated radiometric microassay can be used in a similar manner to quantitate growth of many other filamentous fungi. MATERIALS AND METHODS
Organism T. mentagrophytes var. granulare (American Type Culture Collection 18748, Rockville, Maryland) was maintained on potato dextrose agar. A suspension of microaleuriospores (spores) for inoculum purposes was prepared according to the technique developed by Reinhardt, Allen, Gunnison & Akers (1974). Briefly, the mycelium of a 2-week-old fungus culture was scraped from an agar plate into 0-15 M saline. Spores were separated from the mycelium by agitation in the presence of glass beads followed by filtration through a glass wool column. The spores were concentrated by centrifugation, washed two times with antibiotic solution (cyclohexamide, chloramphenicol, chlorotetracycline), resuspended in distilled water and stored at 4°C. Spore concentration was determined by direct count and spore viability was determined by the dilution plating method.
Radioisotope Uniformly labeled 14C glucose (a4C(U) glucose) with a specific activity of 4-06 mCi/mmole (New England Nuclear Corp., Boston, Mass.) was diluted with distilled water to a concentration of 1-5/ICi/ml. Unlabeled glucose was then added to the radioactive solution to achieve a final concentration of 1-4 x 10- 3 M glucose. This represents a 0-025 ~ glucose solution.
Media The fungi were grown in 0-8 ~ Bacto Nutrient Broth (Difco Laboratories, Detroit, Mich.). The radioactive glucose solution was added to the broth as specified below.
Microculture Technique Spores were cultured in Microtest II microculture plates 3040 (Falcon Co., Oxnard, Calif.) fitted with 3041 lids (Falcon). Each well contained 200/d of nutrient
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broth, 20/B (0-03 #Ci) glucose solution and 20 pl (2 x 104 spores) of spore suspension. The plates were incubated at 34°C. At the desired stage of growth, the mycelial content of each well was collected on filter paper (Grade 934 AH, Reeve Angel, Clifton, N.J.), and washed 10 times with 300 #l of distilled water, 10 times with 300 #I of 5 ~o trichloroacetic acid (TCA) and 10 times with 300 #l volumes of absolute methanol using a multiple automated sample harvester (MASH) (Otto Hiller, Inc., Madison, Wis.). The filter strips were dried at 100°C. Planchets, containing the dried fungal material, were removed and placed into plastic vials containing 2 ml of scintillation cocktail (1 g P O P O P ( 1, 4- his-2- (5-phenyl oxazoly!)-benzene) ; 18g PPO (2, 5-diphenyl-oxazole); in 3 1 of toluene). All samples were subjected to liquid scintillation counting using a Packard Model 3330 spectrometer.
Macroculture Technique Macrocultures of r. mentagrophyteswere set up and harvested in a manner designed to duplicate conditions of the microculture technique. Spores were cultured in 3033 tissue culture tubes (Falcon). Each tube contained 5 ml of nutrient broth, 0-5 ml (0"75 pCi) glucose solution and 0.5 ml (5 x 10 s spores) of spore suspension. The tubes were incubated at 34°C. All cultures were harvested and washed sequentially with 12 ml of distilled water, 12 ml 5 ~ T C A and 12 ml methanol using a 3025 Sampling Manifold (Millipore Corp., Bedford, Mass.) equipped with 0"5 micron pore size cellotate membrane filters (Millipore Corp.). Filters were removed from the sampling manifold, dried at 100°C and placed in glass vials containing 10 ml of Aquasol Universal L.S.C. cocktail (New England Nuclear) and subjected to scintillation counting. Filtrates from macrocultures were collected in the sampling manifold and subjected to scintillation counting. Other macrocultures were extracted by a modification of the method of Gupta & Pramer (1970). The cultures were treated with precooled 20 ~ T C A and shaken at 4°C for 24 h. The fungal mats were then isolated using the sampling manifold and prepared for scintillation counting as described above. The 20 ~ TCA filtrates from the extracted macrocultures were also collected in the manifold. Two ml samples of the filtrates were dialyzed at 4°C against 500 ml of distilled water for 48 h. T h e distribution of radioactivity across the membrane of the dialysis bag (retained molecules with a molecular weight of approximately 10,000 or more) was determined by scintillation counting.
Antifunsal Agent Tolnaftate USP (m, N-dimethylthiocarbanilic acid 0-2 naphthyl ester, Schering Corp., Kenilworth, N.J.) was dissolved in 70 ~ ethanol at a concentration of 400 #g/ ml. Tenfold serial dilutions of this stock were prepared using 70 ~o ethanol. Ten or twenty/A of the appropriate dilutions were pipetted into microculture wells as desired.
Photomicrography Photomicrographs of fungal growth in the microculture wells at various time intervals were prepared using a Nikon inverted microscope equipped with a 35 m m camera.
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RESULTS
Gross observation of growing cultures o f T. mentagrophytes reveals an increasing turbidity with time reflective of the increase in fungal mass. T o determine whether the incorporation of 14C(U) glucose can be used to follow this increasing mass, it is necessary to characterize the relationship between age of culture and the quantity of incorporated label at conditions which permit good growth and at conditions which inhibit growth. Under favorable conditions, if incorporation directly reflects growth, a lag, exponential and stationary phase of incorporation should be definable. Addition of an antifungal agent, tolnaftate, should inhibit incorporation and the subsequent expression of these phases. Using the microculture technique described herein, cultures without tolnaftate and with totnaftate (32 #g/ml of media) were incubated at 34°C and harvested at various intervals up to 9 days. The results of this experiment are graphically depicted in Figure 1. I t can be seen that the incorporation of saC(U) glucose is separable into 3 distinct phases: (1) an initial lag phase (0-8 h) where only a small amount of label is incorporated (50-300 cpm), (2) an exponential phase (8-24 h) where incorporation rapidly increases with time, and (3) a stationary phase (24-96 h) where little or no change in the amount of incorporation is detectable. There was no significant decline in the level of incorporation achieved in this stationary phase over an additional 5-day period. As expected, tolnaftate completely inhibited incorporation at all time intervals examined. I n order to establish more directly the relationship between the incorporation of i4C(U) glucose and increasing mycelial mass, representative photomicrographs
105
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104
~
ATE
OJ t-Z
0
,
LO ca (/) I-Z 0 0 10 2
l 4
I 12
I 24
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I 48
I 72 TIME
l 96 (HOURS)
Figure 1.--The effect of the age of culture on the incorporation of 14C(U) glucose by T. mentagrophytes at 34°C in the absence and preserice of tolnafmte. For those cultures receiving no tolnaftate, each point represents the mean of 9 replicates and is bracketed by the standard error. Tolnaftate-treated cultures were established in duplicate.
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were prepared for each time interval examined above. As can be seen in Figure 2a-f, fungal growth was directly proportional to the incorporation of 14C(U) glucose. At 4 h (fig. 2a) germination had not yet begun and incorporation was only slightly above background (55 cpm). Germination was detectable by 6 h at which time incorporation had increased to 231 cpm (fig. 2b). H y p h a l extension was readily observable at 12 and 16 h (fig. 2c, 2d) and incorporation had increased to 1070 c p m and 3334 c p m respectively. A well-defined mycelium was observable at 24 h (17417 cpm) (fig. 2e) and became significantly denser at 36 h (30674 cpm) (fig. 2f). There was no significant change in the appearance or the level of incorporation at later intervals. Contrastingly, no germination was observable and essentially no incorporation of 14C(U) glucose was detectable in microcultures containing tolnaftate at 32 #g/ml of medium. As additional evidence that the incorporation of a4C(U) glucose can be used to follow" the growth of T . mentagrophytes, the distribution of label between the medium and TCA-soluble and TCA-insoluble fractions of the mycelia at 12, 24 and 36 h in macroculture was determined. The extraction with T C A was designed to separate the soluble radioactive molecules within the fungus (TCA-soluble) from those incorporated into structural macromolecules (TCA-insoluble). Attempts to separate soluble radioactive molecules from insoluble macromolecules with 5 ~ cold T C A failed because of the resistance of the mycelium. To successfully perform the extraction more vigorous conditions were used (incubation of mycelia with precooled 2 0 ~ TCA). The amount of soluble material within the mycelia was determined by subtracting the amount of radioactivity contained in mycelia after exposure to cold 20 ~ T C A from that contained in mycelia not exposed to TCA. Dialysis of the 20 ~ T C A solutions isolated from the mycelia after treatment revealed that 97 ~ or more of the radioactivity contained in the dialysis bag penetrated the membrane. This suggests that the 20 ~ T C A solution was removing only the soluble and small molecular weight molecules from the mycelium and leaving its macromolecular structure intact. As can be seen in Table I, there is a loss of radioactivity from the media with increase in culture time. Correspondingly, there is an increase in the fungus-associated radioactivity. I t has been previously established that such an increase in radioactivity parallels growth of the organism (Fig. 2a-2f). The proportionate amount of the radioTABLE 1.--THE PROPORTIONATE DISTRIBUTION OF 14C(U) GLUCOSE IN CULTURES OF T,
mentagrophytes*
Mycelia TCA~oNb~ •Time in culture (hour's)
Media CPM**
12 24 36
1,252,680er 762,570 369,420
Total CPM
CPM
21,956 6,188 354,895 87,925 789,654 172,653
TCA-I~oNb#
% MyceIia total
CPM
% Mycelia total
28 25 28
15,768 266,970 6t7,001
72 75 72
Total recoverable CPM
1,274,636 1,081,465 1,159,074
*Cultures were established and harvested at the indicated time interval using the macroculture technique as described under Materials and Methods. **Counts per minute. "~Each value is a mean derived from 4 replicate cultures.
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292
Figure 2 . - - T . mentagrophytes at the indicated length of time in culture in the presence of 14C(U) glucose. a) 4 h × 200 b) 6 h x 200
X X X
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QUALMAN JONES AND ARTIS
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label contained in the TCA-soluble and insoluble fractions of the mycelium (approximately 27 ~o and 73 ~ respectively) did not change with the increase in fungal mass. Because the total amount of incorporated label increased with fungal growth and the bulk of this label (73 ~ ) was bound into the macromolecular structure of the organism, incorporation of 14C (U) glucose was concluded to be an accurate monitor of changes in fungal mass. To demonstrate the potential usefulness of the assay, the growth inhibitory activity of tolnaftate, as reflected by its effect upon the incorporation of 14C(U) glucose by T. mentagrophytes, was characterized. Figure 3 depicts the results of this experiment. It can be seen that as the concentration of drug decreases, the amount of radioactive glucose incorporated by the fungus increases proportionately until the inhibitory activity of the drug is lost. Only minimal inhibitory activity was detected at concentrations less than 0.016 yg/ml'. The validity of this putative growth inhibitory activity was confirmed by microscopic examination, which revealed a corresponding decrease in fungal mass with increasing tolnaftate concentration. DIscussioN The present study has demonstrated that an automated microassay employing incorporation of 14C(U) glucose can be used to accurately follow the in vitro growth of the dermatophyte fungus T. mentagrophytes. Figure 1 which plots the amount of label incorporated against time clearly establishes a lag, exponential and stationary phase of fungal growth similar to that described by Swanson & Stock (1966). Gross
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"r
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z rr"
ILl 0..
I0 4
CO t--Z O
10 5
I 0.00016
I 0.0016
I 0.016 TOLNAFTATE
I 0.16
I 1.6
I 16.0
(MICROGRAMS PER ML OF MEDIUM)
Figure 3.--The effect of increasing amounts of tolnaftate on the incorporation of t*C(U) glucose by T. mentagrophytesafter 36h of culture at 34°C. Each point represents the mean of 4 replicate cultures and is bracketed by the standard error.
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estimates of turbidity and the photomicrographs of Figure 2 verify that the incorporation of ~4C(U) glucose accurately depicts these phases. As noted earlier, the total carbohydrate content of T. mentagrophytes has been shown to closely parallel changes in mycelial weight (Swanson & Stock, 1966), as has the neutral sugar content of 7". mentagrophytes (Noguchi et al., 1975). Noguchi et al., (1975) have also reported that 69-5 ~o of the T. mentagrophytes cell wall is composed of sugar. It is not surprising, therefore, that the incorporation of 14C(U) glucose would reflect growth. The above experimenters used 1-4 ~ glucose-supplemented medias. We added a much smaller amount of glucose (a 0-025 ~ solution) to our medium because of the low specific activity of the ~4C(U) glucose; too much unlabeled glucose would have decreased the uptake of radioactive glucose by the fungus. The exact fate of the 14C(U) glucose incorporated by the fungus was not determined by our study. The data accumulated, however, strongly suggest that much of the radio-label is bound within the cell wall of the fungus. Swanson & Stock (1966) concluded that much of the carbohydrate contained in the mycelia of T. mentagrophytes is necessary to maintain the structural integrity of the cell wall. This conclusion was based on the finding that all compounds within the fungus that were studied decreased during starvation (6 days culture time) with the exception of carbohydrate. Decrease in lipid content of the fungus was particularly pronounced during starvation and it was felt this may be the principle energy reserve of the fungus as opposed to storage polysaccharides. It was also noted that the rate of carbohydrate synthesis by T. mentagrophytes was relatively uniform during its growth cycle. Our study also revealed that during starvation (6-9 days in microculture) the amount of incorporated glucose contained within the fungus did not significantly decrease from the level achieved when the fungus first entered the stationary growth phase. Moreover, extraction of mycelia with 20 ~ TCA revealed that at the growth periods studied, the percentages of the radio-label contained in the TCA-soluble (27 ~ ) and TCA-insoluble (73 ~o) mycelial fractions stayed relatively constant. Our observations in combination with the previous work of Swanson & Stock (1966) suggest that T. mentagrophytes utilizes the I*C(U) glucose primarily in the production of TCA-insoluble macromolecules which are likely to be structural components of the fungal cell wall. Prolonged culture time does not seem to effect the breakdown of these structural macromolecules. Whatever amount of glucose is utilized for energy by the fungus appears to be small in comparison to that utilized in macromolecular synthesis. This emphasis on macromolecular synthesis seems to predominate even at the lower amount of glucose contained in our culture medium. This may be due to the large quantities of carbohydrates needed in celI walt synthesis. Since the growth of T. mentagrophytes, like other filamentous fungi, occurs by extension of hyphal tips which involves continual cell wall synthesis, the appropriateness of 14C(U) glucose incorporation as a monitor of growth is evident. We have also used the radio-labels 14C (U) leucine and (all) thymidine in attempts to monitor the growth of T. mentagrophytes. Under the conditions of this study, radioactive leucine and thymidine were inferior to 14C(U) glucose as measures of fungal growth (unreported data). In fact, tritiated thymidine incorporation correlated poorly with gross and microscopic estimates of fungat growth. The sensitivity of the microassay using 14C(U) glucose as a means to monitor growth is demonstrated by the tolnaftate dose response curve (fig. 3). A concentration of the antifungal agent as small as 0-16 pg/ml inhibited incorporation by 50 ~ . The
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Q.UALMAN, JONES AND ARTIS
consistently n a r r o w s t a n d a r d error (approximately 8 ~ ) is indicative of the a c c u r a c y of this technique. A d v a n t a g e s of the r a d i o m e t r i c microassay other t h a n sensitivity a n d a c c u r a c y a r e : (I) it eliminates the a r b i t r a r y n a t u r e of visual estimations of g r o w t h ; (2) its use of a u t o m a t e d harvesting e q u i p m e n t frees it from the b u l k of work associated with visual a n d dry weight d e t e r m i n a t i o n s of g r o w t h ; ( 3 ) i t s use of a radioisotope frees it from the prolonged culture times (1 or 2 weeks c o m p a r e d to 24-36 h with the microassay) necessitated b y visual a n d d r y weight d e t e r m i n a t i o n s of g r o w t h ; (4) it permits the r a p i d performance of complex, m u l t i v a r i a b l e experiments since several h u n d r e d replicate cultures c a n be processed per day. Perhaps a n even greater v a l u e of th e assay lies in its a d a p t a b i l i t y for use i n the study of the growth of n u m e r o u s other filamentous fungi. F u r t h e r work i n our l a b o r a tory has d e m o n s t r a t e d this a u t o m a t e d r a d i o m e t r i c microassay to be a sensitive i n d i c a t o r of the growth of the d e r m a t o p h y t e s Trichophyton rubrum, Epidermophyton floccosum a n d Microsporum canis. This is not surprising since 13 different d e r m a t o p h y t e s are k n o w n to have cell walls composed of 50 ~ sugar or more (No.~uchi et al., 1975). W e have also f o u n d the assay to be a sensitive i n d i c a t o r of the growth of Aspergillus niger a n d Penicillium species. ZUSAMMENFASSUNG Es wird ein amomatisches Mikroverfahren fiir das Wachstum von Trichophyton mentagrophyles und anderen faserartigen Pilzen besehrieben. Das Verfahren bertihrt auf der Verbindung yon I'tC(U)Glukose mit dem Organismus. Einzeluntersuchungen zeigen, dass 73% yon 14C(U)-Glukose in Trichloroacetics/iure unl6sbaren makromoleeularen Bestandteilen des Myceliums gefunden wlrd. D~e Verbindung mit 14C(U)-Glukose stand in direkter Bezlehung zu dem Wachstum das durch die Trtihung visuel und durch Photomikographie bewertet wurde. Die Verhindung mit a4C(U)-Glukose zeigte eine Verz6gerung, exponentielle Und station~re Phase oder eine Plateauphase des Wachstums. Diese Phasen konnten dutch den antifungalen Wirkstoff Tolnaftate vollkommen gehemmt werden. Daraus wurde der Schluss gezogen, dass das Wachstum der faserartigen Pilze erfolgreich mit der beschriebenen radiometrischen Methode gemessen werden kann. Dartiberhinaus ist diese Methode sensitiv, genau, reproduzierbar und schnell durchzuftihren sowie unabhiingig yon den variablen Gr6ssen frtiherer Wachstumsmessungen. ACKNOWLEDGEMEN'IS Support for this study was provided by a Brown-Hazen grant for research in mycology. Expert technical assistance was provided by Sandra Hannaford and Roy Bolles. The advice of Dr. Robert D. King was also appreciated. REFERENCES
BENITEZ,P., MEDOFF,G. & KOBAYASttI,G. S. (1974). Rapid radiometric method of testing susceptibility of mycohacteria and slow-growing fungi to antimicrobial agents. Antimicrobial Agents and
Chemotherapy,6, 29-33. GUPTA, R. K. & PRAMER, D. (1970). Amino acid transport by the filamentous fungus Arthrobot~.ys conoides.Journal of Bacteriology, 103, 120-130. KOBAYASm,G. S., MEDOFF,G., SCHLESSINOER,D., KwAr% C. N. & MUSSaR,W. E. (1972). Amphotericin B potentiation of rifamplcin as an antifungal agent against the yeast phase of tIistoplasrna capsulatum.Science,177, 709-710. Nocucm, T., BANJO,Y., WATANAB~,T., NOZAWA,Y. & ITO, Y. (1975). Carbohydrate composition of the isolated cell walls of dermatophytes. Mycopathologia,55, 71-76. OLOFtNOBA,M. O. (1973). Uptake of 14C(U) glucose by Bot~odiplodiatheobromaePAT and changes in its carbohydrate content during growth. Zeitschriftfur AltgemeineMikrobiologie, 13, 507-5 I5. R~IN~AgDT, J. H., ALL~N, A. M., GUNNISON,D. & AKERS, W. A. (1974). Experimental human Trichophytonmentagrophytesinfections.Journal of IrwestigallveDermatology,63, 419-422.
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STANIER,R. Y., DOr.rDOROVV,M. & ADELBER.O,E. A. (1963). The ~lierobial World, (2nd. Ed.) Engle-
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wood Cliffs, New Jersey: Prentice-Hall, Inc., pgs. 321-341. Swanson, R. & Stock, J. J. (1966). Biochemical alterations of dermatophytes during growth. Applied Microbiology, 14, 438-444.
QUALMAN,H.
E. JONES and W. M. ARTIS
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*Department of Dermatology, Immunodermatology Laboratory, Kresge H R4010 University of Michigan Medical Center, Ann Arbor, Mich. 48109 An automated radiometric microassay of the growth of Trichophytonmentagrophytesand other filamentous fungi is described. The assay is based upon the incorporation of 14C(U) glucose into the organism. Fractionation studies indicate that 73% of the label is found in trichloroacetic acid-insoluble macromolecular components of the mycelium. Incorporation of label directly correlated with growth as estimated by visual scoring of turbidity and as recorded in photomicrographs. Incorporation of 14C(U) glucose delineated a lag, exponential and stationary or plateau phase of growth. These phases could be completely inhibited by the antifungal agent toluaftate. It was concluded that the growth of filamentous fungi can be successfully monitored by the radiometric method described. Moreover, this method is sensitive, accurate, reproducible, rapid and free of the variability inherent in many traditional estimates of growth. For unicellular organisms like bacteria, an increase in population size is an accepted functional definition of growth (Stanier, D o u d o r o f f & Adelberg, 1963). Changes in population size can be measured b y direct counting and m a y be estimated b y culture turbidity or dilution plating techniques. G r o w t h of fungi, because it occurs b y apical h y p h a l extension in filamentous forms and b u d d i n g in the yeast forms, is not definable by a n increase in population size. I t is limited to a measurement of mass. Direct m e a s u r e m e n t o f mass (e.g., d r y weight deternaination) is an accurate b u t a w k w a r d a n d troublesome procedure. Most conventional measurements of fungal growth, therefore, are based u p o n indirect estimates of mass such as colony size or culture turbidity. These estimates are frequently visual and, as such, inherently subjective since growth m a y v a r y with the eye and bias of the observer. These techniques also necessitate prolonged culture of the organism due to their dependence on visible growth. (Benitez, M e d o f f & K o b a yashi, 1974). Reports have appeared in the literature in which the in vitro growth of fungi was monitored via changes in the q u a n t i t y of a structural c o m p o n e n t or in the binding of a radio-labeled precursor. Benitez et al., (1974) and Kobayashi, Medoff, Schlessinger, K w a n & Musser (1972) used the incorporation of radioactive amino acids into proteins, a n d radioactive nucleotides into ribonucleic acid as a measurement of viability of the yeast forms ofHistoplasma capsulatum and Blastomyces dermatitidis which might imply a measu r e m e n t of growth. Swanson & Stock (I966) observed that both the total c a r b o h y d r a t e and the total protein content of T. mentagrophytes var. quinckeanum closely paralleled changes in its mycelial weight. Noguchi, Banno, W a t a n a b e , N o z a w a & Ito (1975) extended this observation by extracting and quantitating the sugar c o m p o n e n t of T. mentagro*Address all correspondence and reprint requests to H. E. Jones, M.D., Division of Dermatology, Emory School of Medicine, Room 215, Woodruff Memorial Building, Atlanta B.A. 30322. 287
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phytes at different times during its growth phase. They found that the content of neutral sugar in the mycelium was directly proportional to mass. Olofinboba (1973) utilized incorporation of 14C(U) glucose by Botyrodiploidia theobromae as a means of following quantitative changes in the carbohydrate content of the fungus during its growth cycle. Changes in the amount of bound glucose were found to reflect changes in the weight of dried mycelium. Because this laboratory is concerned with factors which can influence the growth of dermatophytic fungi, we felt the necessity of developing a sensitive technique to accurately monitor fungal growth. The above observations suggested to us that growth of the pathogenic fungus T. mentagrophytes could be followed by an assay based on the assumption that a radio-labeled sugar would be incorporated into mycelial structural macromolecules. This paper describes an automated microassay of the growth of 7". mentagrophytes var. granulare. It is based upon the incorporation of 14C(U) glucose into trichloroacetic acid (TCA)-insoluble mycelial material. To our knowledge, this is the first report of any attempt to measure dermatophyte growth using radiometric techniques. The utility of this technique, however, is not limited to quantitation of growth of T. mentagrophytes. We also have found that this automated radiometric microassay can be used in a similar manner to quantitate growth of many other filamentous fungi. MATERIALS AND METHODS
Organism T. mentagrophytes var. granulare (American Type Culture Collection 18748, Rockville, Maryland) was maintained on potato dextrose agar. A suspension of microaleuriospores (spores) for inoculum purposes was prepared according to the technique developed by Reinhardt, Allen, Gunnison & Akers (1974). Briefly, the mycelium of a 2-week-old fungus culture was scraped from an agar plate into 0-15 M saline. Spores were separated from the mycelium by agitation in the presence of glass beads followed by filtration through a glass wool column. The spores were concentrated by centrifugation, washed two times with antibiotic solution (cyclohexamide, chloramphenicol, chlorotetracycline), resuspended in distilled water and stored at 4°C. Spore concentration was determined by direct count and spore viability was determined by the dilution plating method.
Radioisotope Uniformly labeled 14C glucose (a4C(U) glucose) with a specific activity of 4-06 mCi/mmole (New England Nuclear Corp., Boston, Mass.) was diluted with distilled water to a concentration of 1-5/ICi/ml. Unlabeled glucose was then added to the radioactive solution to achieve a final concentration of 1-4 x 10- 3 M glucose. This represents a 0-025 ~ glucose solution.
Media The fungi were grown in 0-8 ~ Bacto Nutrient Broth (Difco Laboratories, Detroit, Mich.). The radioactive glucose solution was added to the broth as specified below.
Microculture Technique Spores were cultured in Microtest II microculture plates 3040 (Falcon Co., Oxnard, Calif.) fitted with 3041 lids (Falcon). Each well contained 200/d of nutrient
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broth, 20/B (0-03 #Ci) glucose solution and 20 pl (2 x 104 spores) of spore suspension. The plates were incubated at 34°C. At the desired stage of growth, the mycelial content of each well was collected on filter paper (Grade 934 AH, Reeve Angel, Clifton, N.J.), and washed 10 times with 300 #l of distilled water, 10 times with 300 #I of 5 ~o trichloroacetic acid (TCA) and 10 times with 300 #l volumes of absolute methanol using a multiple automated sample harvester (MASH) (Otto Hiller, Inc., Madison, Wis.). The filter strips were dried at 100°C. Planchets, containing the dried fungal material, were removed and placed into plastic vials containing 2 ml of scintillation cocktail (1 g P O P O P ( 1, 4- his-2- (5-phenyl oxazoly!)-benzene) ; 18g PPO (2, 5-diphenyl-oxazole); in 3 1 of toluene). All samples were subjected to liquid scintillation counting using a Packard Model 3330 spectrometer.
Macroculture Technique Macrocultures of r. mentagrophyteswere set up and harvested in a manner designed to duplicate conditions of the microculture technique. Spores were cultured in 3033 tissue culture tubes (Falcon). Each tube contained 5 ml of nutrient broth, 0-5 ml (0"75 pCi) glucose solution and 0.5 ml (5 x 10 s spores) of spore suspension. The tubes were incubated at 34°C. All cultures were harvested and washed sequentially with 12 ml of distilled water, 12 ml 5 ~ T C A and 12 ml methanol using a 3025 Sampling Manifold (Millipore Corp., Bedford, Mass.) equipped with 0"5 micron pore size cellotate membrane filters (Millipore Corp.). Filters were removed from the sampling manifold, dried at 100°C and placed in glass vials containing 10 ml of Aquasol Universal L.S.C. cocktail (New England Nuclear) and subjected to scintillation counting. Filtrates from macrocultures were collected in the sampling manifold and subjected to scintillation counting. Other macrocultures were extracted by a modification of the method of Gupta & Pramer (1970). The cultures were treated with precooled 20 ~ T C A and shaken at 4°C for 24 h. The fungal mats were then isolated using the sampling manifold and prepared for scintillation counting as described above. The 20 ~ TCA filtrates from the extracted macrocultures were also collected in the manifold. Two ml samples of the filtrates were dialyzed at 4°C against 500 ml of distilled water for 48 h. T h e distribution of radioactivity across the membrane of the dialysis bag (retained molecules with a molecular weight of approximately 10,000 or more) was determined by scintillation counting.
Antifunsal Agent Tolnaftate USP (m, N-dimethylthiocarbanilic acid 0-2 naphthyl ester, Schering Corp., Kenilworth, N.J.) was dissolved in 70 ~ ethanol at a concentration of 400 #g/ ml. Tenfold serial dilutions of this stock were prepared using 70 ~o ethanol. Ten or twenty/A of the appropriate dilutions were pipetted into microculture wells as desired.
Photomicrography Photomicrographs of fungal growth in the microculture wells at various time intervals were prepared using a Nikon inverted microscope equipped with a 35 m m camera.
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RESULTS
Gross observation of growing cultures o f T. mentagrophytes reveals an increasing turbidity with time reflective of the increase in fungal mass. T o determine whether the incorporation of 14C(U) glucose can be used to follow this increasing mass, it is necessary to characterize the relationship between age of culture and the quantity of incorporated label at conditions which permit good growth and at conditions which inhibit growth. Under favorable conditions, if incorporation directly reflects growth, a lag, exponential and stationary phase of incorporation should be definable. Addition of an antifungal agent, tolnaftate, should inhibit incorporation and the subsequent expression of these phases. Using the microculture technique described herein, cultures without tolnaftate and with totnaftate (32 #g/ml of media) were incubated at 34°C and harvested at various intervals up to 9 days. The results of this experiment are graphically depicted in Figure 1. I t can be seen that the incorporation of saC(U) glucose is separable into 3 distinct phases: (1) an initial lag phase (0-8 h) where only a small amount of label is incorporated (50-300 cpm), (2) an exponential phase (8-24 h) where incorporation rapidly increases with time, and (3) a stationary phase (24-96 h) where little or no change in the amount of incorporation is detectable. There was no significant decline in the level of incorporation achieved in this stationary phase over an additional 5-day period. As expected, tolnaftate completely inhibited incorporation at all time intervals examined. I n order to establish more directly the relationship between the incorporation of i4C(U) glucose and increasing mycelial mass, representative photomicrographs
105
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ATE
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,
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Figure 1.--The effect of the age of culture on the incorporation of 14C(U) glucose by T. mentagrophytes at 34°C in the absence and preserice of tolnafmte. For those cultures receiving no tolnaftate, each point represents the mean of 9 replicates and is bracketed by the standard error. Tolnaftate-treated cultures were established in duplicate.
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were prepared for each time interval examined above. As can be seen in Figure 2a-f, fungal growth was directly proportional to the incorporation of 14C(U) glucose. At 4 h (fig. 2a) germination had not yet begun and incorporation was only slightly above background (55 cpm). Germination was detectable by 6 h at which time incorporation had increased to 231 cpm (fig. 2b). H y p h a l extension was readily observable at 12 and 16 h (fig. 2c, 2d) and incorporation had increased to 1070 c p m and 3334 c p m respectively. A well-defined mycelium was observable at 24 h (17417 cpm) (fig. 2e) and became significantly denser at 36 h (30674 cpm) (fig. 2f). There was no significant change in the appearance or the level of incorporation at later intervals. Contrastingly, no germination was observable and essentially no incorporation of 14C(U) glucose was detectable in microcultures containing tolnaftate at 32 #g/ml of medium. As additional evidence that the incorporation of a4C(U) glucose can be used to follow" the growth of T . mentagrophytes, the distribution of label between the medium and TCA-soluble and TCA-insoluble fractions of the mycelia at 12, 24 and 36 h in macroculture was determined. The extraction with T C A was designed to separate the soluble radioactive molecules within the fungus (TCA-soluble) from those incorporated into structural macromolecules (TCA-insoluble). Attempts to separate soluble radioactive molecules from insoluble macromolecules with 5 ~ cold T C A failed because of the resistance of the mycelium. To successfully perform the extraction more vigorous conditions were used (incubation of mycelia with precooled 2 0 ~ TCA). The amount of soluble material within the mycelia was determined by subtracting the amount of radioactivity contained in mycelia after exposure to cold 20 ~ T C A from that contained in mycelia not exposed to TCA. Dialysis of the 20 ~ T C A solutions isolated from the mycelia after treatment revealed that 97 ~ or more of the radioactivity contained in the dialysis bag penetrated the membrane. This suggests that the 20 ~ T C A solution was removing only the soluble and small molecular weight molecules from the mycelium and leaving its macromolecular structure intact. As can be seen in Table I, there is a loss of radioactivity from the media with increase in culture time. Correspondingly, there is an increase in the fungus-associated radioactivity. I t has been previously established that such an increase in radioactivity parallels growth of the organism (Fig. 2a-2f). The proportionate amount of the radioTABLE 1.--THE PROPORTIONATE DISTRIBUTION OF 14C(U) GLUCOSE IN CULTURES OF T,
mentagrophytes*
Mycelia TCA~oNb~ •Time in culture (hour's)
Media CPM**
12 24 36
1,252,680er 762,570 369,420
Total CPM
CPM
21,956 6,188 354,895 87,925 789,654 172,653
TCA-I~oNb#
% MyceIia total
CPM
% Mycelia total
28 25 28
15,768 266,970 6t7,001
72 75 72
Total recoverable CPM
1,274,636 1,081,465 1,159,074
*Cultures were established and harvested at the indicated time interval using the macroculture technique as described under Materials and Methods. **Counts per minute. "~Each value is a mean derived from 4 replicate cultures.
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292
Figure 2 . - - T . mentagrophytes at the indicated length of time in culture in the presence of 14C(U) glucose. a) 4 h × 200 b) 6 h x 200
X X X
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QUALMAN JONES AND ARTIS
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label contained in the TCA-soluble and insoluble fractions of the mycelium (approximately 27 ~o and 73 ~ respectively) did not change with the increase in fungal mass. Because the total amount of incorporated label increased with fungal growth and the bulk of this label (73 ~ ) was bound into the macromolecular structure of the organism, incorporation of 14C (U) glucose was concluded to be an accurate monitor of changes in fungal mass. To demonstrate the potential usefulness of the assay, the growth inhibitory activity of tolnaftate, as reflected by its effect upon the incorporation of 14C(U) glucose by T. mentagrophytes, was characterized. Figure 3 depicts the results of this experiment. It can be seen that as the concentration of drug decreases, the amount of radioactive glucose incorporated by the fungus increases proportionately until the inhibitory activity of the drug is lost. Only minimal inhibitory activity was detected at concentrations less than 0.016 yg/ml'. The validity of this putative growth inhibitory activity was confirmed by microscopic examination, which revealed a corresponding decrease in fungal mass with increasing tolnaftate concentration. DIscussioN The present study has demonstrated that an automated microassay employing incorporation of 14C(U) glucose can be used to accurately follow the in vitro growth of the dermatophyte fungus T. mentagrophytes. Figure 1 which plots the amount of label incorporated against time clearly establishes a lag, exponential and stationary phase of fungal growth similar to that described by Swanson & Stock (1966). Gross
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10 5
I 0.00016
I 0.0016
I 0.016 TOLNAFTATE
I 0.16
I 1.6
I 16.0
(MICROGRAMS PER ML OF MEDIUM)
Figure 3.--The effect of increasing amounts of tolnaftate on the incorporation of t*C(U) glucose by T. mentagrophytesafter 36h of culture at 34°C. Each point represents the mean of 4 replicate cultures and is bracketed by the standard error.
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estimates of turbidity and the photomicrographs of Figure 2 verify that the incorporation of ~4C(U) glucose accurately depicts these phases. As noted earlier, the total carbohydrate content of T. mentagrophytes has been shown to closely parallel changes in mycelial weight (Swanson & Stock, 1966), as has the neutral sugar content of 7". mentagrophytes (Noguchi et al., 1975). Noguchi et al., (1975) have also reported that 69-5 ~o of the T. mentagrophytes cell wall is composed of sugar. It is not surprising, therefore, that the incorporation of 14C(U) glucose would reflect growth. The above experimenters used 1-4 ~ glucose-supplemented medias. We added a much smaller amount of glucose (a 0-025 ~ solution) to our medium because of the low specific activity of the ~4C(U) glucose; too much unlabeled glucose would have decreased the uptake of radioactive glucose by the fungus. The exact fate of the 14C(U) glucose incorporated by the fungus was not determined by our study. The data accumulated, however, strongly suggest that much of the radio-label is bound within the cell wall of the fungus. Swanson & Stock (1966) concluded that much of the carbohydrate contained in the mycelia of T. mentagrophytes is necessary to maintain the structural integrity of the cell wall. This conclusion was based on the finding that all compounds within the fungus that were studied decreased during starvation (6 days culture time) with the exception of carbohydrate. Decrease in lipid content of the fungus was particularly pronounced during starvation and it was felt this may be the principle energy reserve of the fungus as opposed to storage polysaccharides. It was also noted that the rate of carbohydrate synthesis by T. mentagrophytes was relatively uniform during its growth cycle. Our study also revealed that during starvation (6-9 days in microculture) the amount of incorporated glucose contained within the fungus did not significantly decrease from the level achieved when the fungus first entered the stationary growth phase. Moreover, extraction of mycelia with 20 ~ TCA revealed that at the growth periods studied, the percentages of the radio-label contained in the TCA-soluble (27 ~ ) and TCA-insoluble (73 ~o) mycelial fractions stayed relatively constant. Our observations in combination with the previous work of Swanson & Stock (1966) suggest that T. mentagrophytes utilizes the I*C(U) glucose primarily in the production of TCA-insoluble macromolecules which are likely to be structural components of the fungal cell wall. Prolonged culture time does not seem to effect the breakdown of these structural macromolecules. Whatever amount of glucose is utilized for energy by the fungus appears to be small in comparison to that utilized in macromolecular synthesis. This emphasis on macromolecular synthesis seems to predominate even at the lower amount of glucose contained in our culture medium. This may be due to the large quantities of carbohydrates needed in celI walt synthesis. Since the growth of T. mentagrophytes, like other filamentous fungi, occurs by extension of hyphal tips which involves continual cell wall synthesis, the appropriateness of 14C(U) glucose incorporation as a monitor of growth is evident. We have also used the radio-labels 14C (U) leucine and (all) thymidine in attempts to monitor the growth of T. mentagrophytes. Under the conditions of this study, radioactive leucine and thymidine were inferior to 14C(U) glucose as measures of fungal growth (unreported data). In fact, tritiated thymidine incorporation correlated poorly with gross and microscopic estimates of fungat growth. The sensitivity of the microassay using 14C(U) glucose as a means to monitor growth is demonstrated by the tolnaftate dose response curve (fig. 3). A concentration of the antifungal agent as small as 0-16 pg/ml inhibited incorporation by 50 ~ . The
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Q.UALMAN, JONES AND ARTIS
consistently n a r r o w s t a n d a r d error (approximately 8 ~ ) is indicative of the a c c u r a c y of this technique. A d v a n t a g e s of the r a d i o m e t r i c microassay other t h a n sensitivity a n d a c c u r a c y a r e : (I) it eliminates the a r b i t r a r y n a t u r e of visual estimations of g r o w t h ; (2) its use of a u t o m a t e d harvesting e q u i p m e n t frees it from the b u l k of work associated with visual a n d dry weight d e t e r m i n a t i o n s of g r o w t h ; ( 3 ) i t s use of a radioisotope frees it from the prolonged culture times (1 or 2 weeks c o m p a r e d to 24-36 h with the microassay) necessitated b y visual a n d d r y weight d e t e r m i n a t i o n s of g r o w t h ; (4) it permits the r a p i d performance of complex, m u l t i v a r i a b l e experiments since several h u n d r e d replicate cultures c a n be processed per day. Perhaps a n even greater v a l u e of th e assay lies in its a d a p t a b i l i t y for use i n the study of the growth of n u m e r o u s other filamentous fungi. F u r t h e r work i n our l a b o r a tory has d e m o n s t r a t e d this a u t o m a t e d r a d i o m e t r i c microassay to be a sensitive i n d i c a t o r of the growth of the d e r m a t o p h y t e s Trichophyton rubrum, Epidermophyton floccosum a n d Microsporum canis. This is not surprising since 13 different d e r m a t o p h y t e s are k n o w n to have cell walls composed of 50 ~ sugar or more (No.~uchi et al., 1975). W e have also f o u n d the assay to be a sensitive i n d i c a t o r of the growth of Aspergillus niger a n d Penicillium species. ZUSAMMENFASSUNG Es wird ein amomatisches Mikroverfahren fiir das Wachstum von Trichophyton mentagrophyles und anderen faserartigen Pilzen besehrieben. Das Verfahren bertihrt auf der Verbindung yon I'tC(U)Glukose mit dem Organismus. Einzeluntersuchungen zeigen, dass 73% yon 14C(U)-Glukose in Trichloroacetics/iure unl6sbaren makromoleeularen Bestandteilen des Myceliums gefunden wlrd. D~e Verbindung mit 14C(U)-Glukose stand in direkter Bezlehung zu dem Wachstum das durch die Trtihung visuel und durch Photomikographie bewertet wurde. Die Verhindung mit a4C(U)-Glukose zeigte eine Verz6gerung, exponentielle Und station~re Phase oder eine Plateauphase des Wachstums. Diese Phasen konnten dutch den antifungalen Wirkstoff Tolnaftate vollkommen gehemmt werden. Daraus wurde der Schluss gezogen, dass das Wachstum der faserartigen Pilze erfolgreich mit der beschriebenen radiometrischen Methode gemessen werden kann. Dartiberhinaus ist diese Methode sensitiv, genau, reproduzierbar und schnell durchzuftihren sowie unabhiingig yon den variablen Gr6ssen frtiherer Wachstumsmessungen. ACKNOWLEDGEMEN'IS Support for this study was provided by a Brown-Hazen grant for research in mycology. Expert technical assistance was provided by Sandra Hannaford and Roy Bolles. The advice of Dr. Robert D. King was also appreciated. REFERENCES
BENITEZ,P., MEDOFF,G. & KOBAYASttI,G. S. (1974). Rapid radiometric method of testing susceptibility of mycohacteria and slow-growing fungi to antimicrobial agents. Antimicrobial Agents and
Chemotherapy,6, 29-33. GUPTA, R. K. & PRAMER, D. (1970). Amino acid transport by the filamentous fungus Arthrobot~.ys conoides.Journal of Bacteriology, 103, 120-130. KOBAYASm,G. S., MEDOFF,G., SCHLESSINOER,D., KwAr% C. N. & MUSSaR,W. E. (1972). Amphotericin B potentiation of rifamplcin as an antifungal agent against the yeast phase of tIistoplasrna capsulatum.Science,177, 709-710. Nocucm, T., BANJO,Y., WATANAB~,T., NOZAWA,Y. & ITO, Y. (1975). Carbohydrate composition of the isolated cell walls of dermatophytes. Mycopathologia,55, 71-76. OLOFtNOBA,M. O. (1973). Uptake of 14C(U) glucose by Bot~odiplodiatheobromaePAT and changes in its carbohydrate content during growth. Zeitschriftfur AltgemeineMikrobiologie, 13, 507-5 I5. R~IN~AgDT, J. H., ALL~N, A. M., GUNNISON,D. & AKERS, W. A. (1974). Experimental human Trichophytonmentagrophytesinfections.Journal of IrwestigallveDermatology,63, 419-422.
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STANIER,R. Y., DOr.rDOROVV,M. & ADELBER.O,E. A. (1963). The ~lierobial World, (2nd. Ed.) Engle-
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wood Cliffs, New Jersey: Prentice-Hall, Inc., pgs. 321-341. Swanson, R. & Stock, J. J. (1966). Biochemical alterations of dermatophytes during growth. Applied Microbiology, 14, 438-444.
