Growth, physiology and ultrastructure of the pathogenic fungus Candida albicans
The growth of the yeast form of C.albicans in Sabouraud dextrose medium is described. Yeast cells divided by multipolar budding and had a generation time of 62 min at 37°C. The generation time as measured by cell number and dry weight was shorter than that measured by cell protein, DNA and culture turbidity suggesting that cells became lighter and contained progressively less protein and DNA as they approached stationary phase culture. The times occupied by budding, mitosis, septation and cell division during cell cycle are given. Newborn calf serum induced germination in all cells within 3 h and could be diluted to 20% (v/v) without reduction in germination. Germination was greatly reduced in serum medium at pH values of less than 5.5 and with inoculation densities of greater than 107 cells/ml. Endogenous chitinase activity in serum could be removed with no loss of ability of the serum to induce morphogenesis. Yeast cells germinated in Sabouraud dextrose broth if cultures were grown at 25°C until they reached stationary phase then inoculated into fresh medium at pH 6.8. Stationary phase cells produced buds at 25°C or at 37°C with a pH of 4.5. The total amino acid pools of cells were reduced during the first hour in both methods of inducing germination. On the basis of these results it is suggested that germination may occur in response to temporary or prolonged starvation. The ultrastructure of the mycelial phase was studied in thin sections of fixed material and in shadow casts of the chemically purified chitinous wall layer. The septum had a 25 nm central micropore which did not allow the passage of cell organelles and consequently delimited single nuclei within mycelial compartments. The growth and development of mycelia in serum-containing media is described. Initially colonies were undifferentiated (all the hyphae in the mycelium had approximately the same diameter, extension rate, apical and intercalary compartment lengths) whereas older mycelia differentiated at the colony margin to produce leading hyphae that were wider, faster growing and had longer apical and intercalary compartments than the branches they subtended. Early colony development exhibited unusual features: germ tube extension was linear (not exponential as in other fungi) and there was a prolonged delay between septation and the onset of branch formation. The subsequent growth and branching patterns were otherwise similar to those of other mycelial moulds. The specific growth rate, mean rate of tip extension, hyphal growth unit and branch and length ratios of mycelia grown on 20% serum agar are given. The septa did not prevent cytoplasmic flow and consequently allowed the peripheral growth zone to span several compartments. During germination of C.albxoans in solid or liquid media the parent yeast cell and then sub-apical regions of the developing germ tube became extensively vacuolated. Intercalary compartments were often almost entirely vacuolated while the apices of germ tubes and branches maintained a high cell solids content. This length of non-vacuolate hypha may correspond to the growth zone of the organism. The hyphae therefore appeared to have sub-apical volumes of protoplasm which migrated forward with their extending apices. These observations may explain the observed unexpected linear growth of these germ tubes and the delay between septation and branch formation during filamentous growth. Chitin microfibrils exposed by chemical extraction of hyphal walls of C.albicans, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidiodes brasiliensis, Coprinus ainereus and Mucor mucedo were of variable morphology but gave similar infrared spectra, X-ray diffraction patterns and behaved as pure chitin in chromatographic analyses. The microfibrils of the four dimorphic fungi that were studied were shorter than those in the moulds C.cinereus and M.mucedo but were similar to those reported for the yeast Saccharomyces cerevisiae. In C.albicans the microfibrils in the septal plates of hyphae were predominantly tangentially orientated and were longer than those in the lateral walls. Microfibrils produced by chitin synthase in vitro were very much longer than any observed from hyphal preparations.