The attenuated total reflectance infrared microspectroscopy of bacterial colonies on agar plates : an honors thesis (HONRS 499)

Abstract

The attenuated total reflectance spectra of 44 species of bacteria were obtained while still on the agar using the infrared microscope. Compared to the specular reflectance spectra of the same species, the v(OH) and S(OH) absorptions are not broad and totallyabsorbing due to the reduced pathlength resulting from using the ATR objective. The ATR spectra show slight frequency shifts in bands from the specular reflectance spectra. These shifts are consistently higher in the ATR spectra, except for the v(P02) band andthe C-O-C and C-O-P stretching bands, which are lower. Principal components regression analysis was used to obtain a measure of differentiating potential of our ATR spectra. A positive one, +1, was assigned to Gram positive species and a negative one, - 1, was assigned to Gram negative species. The prediction results after performing a cross validation show that Gram positive bacteria have positive predicted values for Gram stain while Gram negative bacteria have negative values. Also, it was found that ATR spectra can be obtained from bacteria grown on more than 1 type of agar and still have good differentiating capabilities for Gram stain using PCR analysis. Overall, using ATR spectra from colonies directly on the agar provides significantly better differentiating spectral information and predicting capabilities than specular reflectance spectra.