The in situ infrared microspectroscopy of bacterial colonies on agar plates

The specular reflectance infrared spectra of thirty species of bacteria were obtained in situ, without their removal from the agar growth media, using the infrared microscope. Compared to transmittance spectra obtained from dehydrated films, the transformed reflectance spectra show significant band shifts which are greater for gram positive bacteria. The most notable shift occurs in the v[a](PO2-) band which shifts 8 cm(-1) higher upon removal of water. In addition, some measure of the differentiation potential of our in situ reflectance spectra was gathered by using principal components regression as a method of discrimination. We assigned a +1 for a gram positive property and a -1 to a gram negative property for each different bacteria species. The prediction results from a cross validation show that all the gram positive bacteria retain positive values for Gram stain predictions while all the gram negative species retain their negative values, suggesting a simple marker for differentiation. Overall, using reflectance spectra from colonies directly on the agar requires no sample preparation and provides slightly better differentiating spectral information than the transmittance spectra of dehydrated films.