FT-IR microspectroscopy for microbiological studies

In this article we present an infrared microspectroscopic investigation on Candida albicans microcolonies, taken as a model system for studies on othe r microorganisms. Excellent Fourier transform infrared (FT-IR) absorption s pectra from 4000 to 850 cm(-1) have been collected in only 20 a from sampli ng areas of 100 x 100 mu m(2) in microcolonies, which had been transferred from the agar plate onto zinc selenide (ZnSe) windows. When different regio ns within a single microcolony were investigated, absorption spectra with i mportant differences in the carbohydrate absorption (from 1200 to 850 cm(-1 )) were detected for the cells in the center and in the periphery of the co lony. Results obtained on microcolonies grown on solid agar with increasing dextrose concentrations indicated that the observed spectral heterogeneity was related to differences in dextrose uptake, which was lower for the old cells in the center of the colony than for the metabolically active cells at the periphery. Although it is otherwise difficult to quantitatively eval uate the dextrose uptake in a microcolony, FT-IR absorption microspectrosco py offers a new and rapid method for the analysis of this process. The poss ibility of studying highly absorbing colonies by attenuated total reflectio n (ATR) by means of an ATR microscope germanium objective is also presented here for the first time. An evaluation of the contact area sampled by this technique is reported with a discussion of the spatial resolution, the qua lity and the potential of the ATR measurements. (C) 2000 Elsevier Science B .V. All rights reserved.