This study presents an improved system that enables isolation of single viable prokaryotic cells from a mixture of cells. The system is based on an inverted microscope, a microinjector and a micromanipulator. The isolated cell is captured in a microcapillary from a volume of 400 mu l and transferred to an appropriate growth medium. Validation of the system was performed using two fluorescent strains: Pseudomonas putida expressing red fluorescent protein (DsRed), and Escherichia coli expressing green fluorescent protein (GFP). A mixture (100:1) of the constructed fluorescent strains was subjected to isolation experiments and nine out of ten individually isolated cells yielded axenic cultures of E. coli. Upon construction and validation, the system was used to isolate and subsequently cultivate axenic cultures of the thermophilic Archaeon Metallosphaera sedula and the hyperthermophilic Archaeon Sulfolobus solfataricus from enriched hot spring samples. The high efficiency of single-cell isolation and cultivation demonstrated over a range of temperatures-90% (30 degrees C), 85% (70 degrees C) and 95% (80 degrees C)-from different environments is probably due to the elimination of osmotic stress and limitation of temperature fluctuations during the isolation process, as a result of the large sample volume from which the cells are isolated.