Growth strategy of the pathogenic yeast Cryptococcus neoformans submerged culture under different cultivation formats.

Growth patterns of Cryptococcus neoformans submerged culture in different culture volumes, intensity of agitation and types of sealing were evaluated to better understand the physiological role of hypoxia response in this yeast. When low intensity agitation was set at high culture volumes and air exchange between the cultivation vessel and external environment was not abolished completely, the cells proliferated slowly but steadily. On the other hand, when the intensity of agitation was high but the vessel was withheld from fresh air supply, the cells first proliferated rapidly, then arrested completely and finally died. Therefore, the central strategy of C. neoformans here seems to lie in its proliferation-rate adjustment to the available oxygen levels and not in its capacity to survive under anoxia. The data support the opinion that the cultures grown under limited aeration (even though not completely withheld from fresh air supply) are much closer to the real cryptococcal life in human tissues than conventional well-aerated exponential cultures.

Peculiar clusters of daughter cells observed in Cryptococcus neoformans grown in sealed microtiter plates.

Cryptococcus neoformans was grown in 96-well microtiter plates sealed by foil which is less than 0.01 % permeable to oxygen. On day 14 of the cultivation, we observed peculiar clusters of small droplike daughter cells arranged around < or = 4 % of mother cells. The fact that most of the other cells had died indicates that few cells had been able to survive hypoxic conditions and escape the cell-cycle arrest. However, their daughters were unable to separate from them and to continue their proliferation under such conditions.

Primer R108 performs best in the RAPD strain typing of three Aspergillus species frequently isolated from patients.

We evaluated the suitability of various primers for the RAPD (random amplified polymorphic DNA) accurate species identification and strain typing of Aspergillus clinical isolates. Five primers described previously were tested for their discriminatory power in three Aspergillus species (A. fumigatus, A. niger agg. and A. flavus - 23 clinical isolates and 2 reference strains). Clustering of RAPD fingerprints corresponded well with the identification based on morphological features. All isolates were resolved as different strains using the primer R108 and the RAPD protocol optimized for a Robocycler thermal cycler. RAPD with the primer R108 thus can be considered to be a valuable, simple and powerful tool for identification and strain delineation of Aspergillus spp.