Each series selleck inhibitor contained at least 40 sections. Sections were viewed and analyzed with a light Axio Imager A1 (Carl Zeiss) microscope; images were captured using an AxioCam (Carl Zeiss) digital camera with the software axiovision ac. Trace software igl trace 1.26b (Fiala, 2002) was used

to adjust serial sections by their contours. The 3D images were exported to WRML format, with final rendering using 3D Studio Max9 (Autodesk). Electron microscopy of the cell surfaces of yeasts grown in oil-containing media revealed profound structural alterations in the cell walls as compared with yeasts grown without hydrocarbons. Depending on the species, the yeasts could be divided into two groups. Group one included several species of Candida, Torulopsis and Shwanniomyces. When grown on either hexadecane, n-alkane mixtures (C12–C20) or crude oil, these yeasts formed ‘canals’ in their cell walls. The canals were numerous, with up to 100 canals per cell, and were especially vivid on the carbon–platinum replicas of the cell surface (Fig. 1a and b). The formation of the canals was substrate-dependent. When cells that had been grown on oil were transferred

to a medium with glucose as a sole carbon source, the canal-forming yeasts reverted to a morphotype without canals (Fig. 1c). Along with canal formation, the yeasts S. occidentalis, T. candida and C. maltosa also secreted copious amounts of fibrilar substances when cultivated in media with hexadecane, a mixture of n-alkanes or crude oil. Ultrathin sections and freeze fraction micrographs vividly click here demonstrated that this exosubstance was anatomically bound with the canal features (Fig. 2a–c). Cytochemical staining Etofibrate of these cells with diaminobenzidine further revealed the presence of oxidative enzymes

that were concentrated in the canals (Fig. 3a and b). The oxidative enzymes could also be observed in canals of partially purified cell wall fractions from these yeasts, suggesting that these enzymes are ionically or covalently bound with these modified sites of the cell wall (Fig. 3c). Immunocytochemical staining (Fig. 3d) further showed that cytochrome P-450 was concentrated in distinct locations within the cell walls. All of these facts confirmed the supposition that primary oxidation of hydrocarbons by yeasts occurs mainly in the canals where degradative enzymes are entrapped in a polymer matrix. In contrast to the canal-forming yeast, a second group of yeasts including C. lipolytica and C. paralipolytica did not form canals when grown in hydrocarbon- or crude oil-containing media, but instead secreted large amounts of fibrilar substances. The carbon–platinum replicas of these yeasts were quite smooth (Fig. 3e) and the yeasts appeared to secrete the fibrilar substances over their entire cell surface. The products of the cytochemical staining reaction targeting oxidative enzymes were located both on the cell surfaces and on the exocellular films (Fig. 3f).