Development of natural culture media for rapid induction of Fonsecaea pedrosoi sclerotic cells in vitro

Abstract

Fonsecaea pedrosoi is the main agent of chromoblastomycosis, a skin disease presenting verrucous lesions, in which round, thick-walled sclerotic cells are found. In vitro induction of sclerotic cells is time-consuming (20 to 45 days) and temperature dependent. We present two new natural media that reduce the sclerotic-cell induction time to only 2 days.

FIG. 1.

Fonsecaea pedrosoi sclerotic cells were obtained after scraping of chromoblastomycosis lesions. The cells gave rise to hyphae and conidia after in vitro culture. (A and B) Skin scrapings were collected and analyzed after clarification with 20% KOH, revealing well-defined, septated, sclerotic cells. (C) Greenish-black colonies grew from culture of this material on Mycosel. (D) Characteristic dematiaceous hyphae originating terminal cylindrical conidiophores with small subhyaline conidia were observed upon microculture. Bars, 4 μm (A), 3 μm (B), 0.5 cm (C), and 10 μm (D).

FIG. 2.

The morphological characteristics of sclerotic cells induced in either of the two natural media are similar. (A and B) Sclerotic cells obtained after culture of conidia in Theobroma grandiflorum or Bactris gasipaes natural media were analyzed by optical microscopy with no special staining. (A) Brownish, multiseptated cells (arrow) were evident. (B) Remnants of conidia adhering to the newly formed sclerotic cells (arrows) were observed. (C) SEM of aggregates of sclerotic cells demonstrates septated division (white arrow) and remnants of conidia (yellow arrow). (D) TEM of a sclerotic cell shows typical thick-walled septation (arrow) and vesicles containing electron-dense material (asterisks). Data are representative of one of three independent experiments, which were performed in triplicate. Bars, 10 μm (A and B), 5 μm (C), and 1 μm (D).