Growth characteristics of the slow-growing actinobacterium Frankia sp. strain CcI3 on solid media

Frankia sp. strains are slow-growing Actinobacteria that form N₂-fixing root nodules on certain angiosperm trees and shrubs. These organisms are generally cultured in liquid media as routine growth on solid media is impractical because of lengthy incubation periods. Liquid media is problematic for filamentous organisms because unicellular spores are difficult to isolate, and mutants are difficult to separate. The goal of this study was to optimize Frankia CcI3 growth on solid media. Starting with an appropriate basal medium containing pyruvate as a carbon source, various peptones were individually tested to determine if the time needed for colonies to appear could be reduced. Bacto™ Proteose Peptone #3 at 1.6 mg ml⁻¹ final concentration promoted the best growth with colonies visible after 3 days, compared with the 7- to 10-day incubation required for control unamended media. Gellan gum gave a more rapid growth response as compared with agar. Increased calcium concentrations were required to promote solidification of gellan gum. Sporulation occurred within 2 weeks of plating CcI3 with colonies changing color from cream to reddish-brown. Scanning and transmission electron micrographs revealed the architecture of colonial development. Colonies developed in a lens-shaped manner mainly by penetration into the gel. Older colonies had numerous empty hyphae with sporangia developing near or at the surface and with crystalline material, presumably pigment, interspersed among the hyphae. Occasional vesicles were seen mixed within the colonies. Colonial development of Frankia CcI3 is reminiscent of that described for other members of the filamentous Suborder Frankineae.