THE C O M P A R A T I V E MORPHOLOGY OF THE F U N G A L MAT AND BALL OF T R I C H O P H Y T O N R U B R U M

by

R. A. ZuSSMAN, E. E. VICHER & I. LYON

(Depts. o/ Microbiol. and Biol. Chem., Univ. o/Illinois Colt. o[ Med., Chicago, Illinois)

(with 3 figs.)

(16.Xli.1960)

Trichophyton rubrum, (CDC strain), seeded onto an agar surface, grows in the form of a slightly convex mat. When the fungus is seeded into a liquid medium and agitated on a rotary shaker, spheroidal clones appear. Freeze-dried specimens of both forms were sectioned, studied and photographed. To our knowledge this con- stitutes the first such s tudy of a dermatophyte; the findings are described in this report.

MATERIALS AND METHODS

Fifteen ml of Nickerson's growth medium (NIcKERSON & CHAD- WlCI¢ (1946)) containing 1.5 °/o agar were dispensed into a sterile Petri dish. After hardening, the medium was centrally inoculated with a loopful of fungal homogenate (VIcHER, LYON & WHITE (1959)) preparedirom a 7 day old culture of T. rubrum and incubated at 30 ° C until the colony size was at least 3 cm (attained in approx- imately 10 days). This same medium, sans agar, as well as a che- micaUy defined medium (Zuss~AN (1959)), was used for the liquid cultures; 135 ml of these media were dispensed into separate sterile 25O ml Erlenmeyer flasks. Each flask was inoculated with 0.1 ml of the fungal homogenate and incubated at room temperature for at least three weeks, with continuous shaking, until well-defined, pigmented clones were observed.

A strip 5 mm wide, cut from tile center of tile mat, was instantly frozen in an isopentane bath immersed in liquid nitrogen. Whole spheroids were treated similarly. The frozen materials were dessi- cated in vacuo for several days and imbedded ill paraffin. Sections, 10 # thick, were mounted on slides and cleared in xylol. Previous studies (ZuSSMAN, LYON & VICHER (1960)) showed that this solvent

FUNGAL MAT AND BALL OF T. RUBRUM 199

F i g . 1. C r o s s - s e c t i o n o f a p i g m e n t e d , t h r e e w e e k o l d f u n g a l m a t . 3 3 0 X .

200 R. A. ZUSSMAN, E. E. VICHER ~L I. LYON

does not dissolve tile pigments of T. rubrum. Photomicrographs were made on Kodak M plates with a green filter for contrast.

RESULTS

Figure I is a cross-section of a mature fungal mat. Seven dis- tinct zones may be seen:

(A) paper used in the preparation of the freeze-dried specimen, (B) submerged, vegetative, non-pigmented mycelia with a few

extracellular bodies containing a purple pigment, (C) a relatively non-compact arrangement of hyphae, a few of

which have yellow wails, ~ ) many closely-packed hyphae with yellow walls and great

numbers of intramural and extracellular melanin-like granules; the hyphae which bear the dark pigment granulae are often enlarg- ed and grossly distorted, resembling "bags full of marbles" (fig. 2),

Fig. 2. Cross-section of a pigmented fungal mat showing characteristic enlarged granule-filled hypha. 1200 ×.

(E) a transition zone in which the semi-compact hyphae contain fewer pigment granules and yellow filaments, with a very few extracellular granules and not many atypical hyphae,

(F) the great mass of the fungM colony, consisting of non-pig- mented, non-reproductive hyphae, and

(G) the uppermost layer composed of hyphae that bear the re- productive structures, chlamydospores and microconidia. Macro-

FUNGAL MAT AND BALL OF T, RUBt{'UNI 2 0 1

F i g . 3. C r o s s - s e c t i o n o f a f u n g a l s p h e r o i d . 3 3 0 × .

202 ~. A. ZUSSMAN, 5E. E.VICHER (~ t . LYON

conidia have not been demonstrated in the CDC strain of T. rubrum used in this study.

The gross morphology of the fungal spheroid is different from that of the mat, but similarities exist on the cellular level. The spheroid (figure 3) is composed of three zones:

(A) an outermost layer which is thin, compact, physically tough and highly pigmented; no pigment granules are found free in the extracellular spaces,

(B) a transitional zone in which about 15 % of the hyphal walls are yellow, and

(C) a watery, non-compact, non-pigmented zone comprising most of the fungal volume. No reproductive structures of any kind have been observed in this, or any other zone of the spheroidal form.

DISCUSSION

Comparison of figures I and 3 reveal both similarities and dis- similarities between the fungal mat and spheroid. There can be little doubt that a morphological relationship exists between struc- tures of zones (D) of the mat and (A) of the spheroidal form, and of (E) and (B), and (F) and (C) of the mat and spheroid, respecti- vely.

From these observations, one may conclude that the spheroid is equivalent to an involuted fungal mat, folded in such a way that the internal portion of the spheroid is analogous to the aerial myce- lia of the mat, and the outer zone of the former to the undersurface of the mat. These structural similarities suggest similarities in function.

The manner in which the mat and spheroidal forms develop is not the same. SeverM days after the inoculation of a Petri dish, vegetative mycelia may be seen in the medium, followed by tufts of white aerial mycelia directly over the point of inoculation; no differentiation into other specialized components is seen at this time.

After several days, the diameter of the colony increases and the central regions appear to be far more dense than the periphery. Shortly thereafter, yellow hyphae appear centrally and extend con- centrically with the passage of time. Later, grotesque, enlarged, granule-bearing hyphae may be seen in the center of the clone. Finally, at morphological matur i ty the spread of the granule-bear- ing zone overtakes new peripheral growth as well as the peripheral boundaries of the yellow hyphae. This results in the fusion of these two zones into a single one which now extends almost to the periph- ery of the clone. In summary, growth, differentiation and maturation proceed from the point of inoculation (the oldest area) to the youngest areas which are the last to differentiate and mature.

FUNGAL MAT AND BALL OF T. RUBRUM 203

In the initial growth period of the spheroidal clone, the mycelia extend from the nidus to form a stellar clone that is centrally dense. When the clone reaches optimal size 1), peripheral growth ceases and is followed by internal growth tending to increase the cellular density of the clone. An early result of this process is the marked, localized thickening of the periphery which causes the clone to lose its early characteristically stellar appearance. Little or no in- crease in clonal size occurs thereafter. Grossly, hard-walled sphe- roids with fluid-saturated centers are seen and intramural pig- mentation begins, terminating in an appearance similar to that seen in figure 3. Further incubation, with or without shaking, is characterized by the release of one or more conical fragments from a majority of the spheroids. The culture becomes a mixture of conical fragments, punctured spheroids and bubbles of gas. The apex of the conical fragment corresponds to the center of the clone, and the base to its periphery. The significance of these morpholo- gical forms and the origin of tile gas bubbles are not known. The latter may merely represent air entrapped during shaking or may be a product of fungal metabolism. That this organism has a mark- ed capacity for reductive metabolism is indicated by the forma- tion of arsine gas in a medium containing arsenate (ZusSMAN, VICHER & LYON (1961).

Other studies (ZIJSSMAN (1959); ZUSSMAN, LYON & VICI~ER (1960)) have shown that several different pigments were produced by the two morphological forms of T. rubrum. Reconciliation of morphological differences with the various classes of pigment produced (e. g., anthraquinoid, melanoid, and perhaps others) remains a problem of considerable magnitude and interest.

SUMMARY

A CDC strain of Trichophyton rubrum was grown on solid medium and in shake cultures. The resulting forms (mat-like and spheroidal, respectively) were prepared for sectioning and study by the freeze- drying technic. The results indicate certain similarities and differ- ences in growth and pigmentation patterns, depending upon the method of culture. Pigmentation is not random but occurs in spe- cific areas of the fungal clone. The relationship between each form and their pigmentation patterns is briefly described and discussed.

References

NICKERSON, W. J. & CttADWtCK, J. ]3. (1946) On the respiration of dermatophytes; Arch. Biochem. 10, Sl--100.

VICHB~, E. E., LYON, I. & WHITe, E. L. (1959) Studies on the respiration of Trichophyton rubrum; Mycopathol. et Mycol. Appl., XI, 185--195.

1) Optimaa size depends upon the number of clones in the medium, flask size, composition of the medium, flask shaking rate, temperature and other factors.

204 R.A. ZUSMSAN, E. E. VICHER & I. LYON

ZtlSSMAN, R. A, (1959) Pigment forming mechanisms of Trichophyton rubrum; Master's thesis, U. of Illinois Graduate School. Chicago, Illinois.

ZUSSMAN, R. A., LYON, I. & VICHER, E. E. (1960) Melanoid pigment production in a strain o5 Trichophyton rubrum; J. Bact. 80, 708--713.

ZUSSMAN, R. A., VICI~ER, E, E. & LYON, I. (1961) Arsine production by a strain of Trichophy on rubrum; J. Bact. 81, 157.