The infrared spectroscopic Gram stain

The attenuated total reflectance (ATR) spectra of bacterial colonies were obtained in situ, without their removal from the agar growth media using the infrared microscope. Principal components regression (PCR) analysis was used to obtain a measure of the differentiating potential of the ATR spectra obtained on two standard sets consisting of 31 and 44 species, respectively, grown on trypticase soy agar. A quantitative response value of +1 was assigned to Gram positive species and -1 was assigned to Gram negative species. In a cross validation experiment, the Gram stain property correlated with the factor loadings with a multiple correlation of 0.9502 and 0.9520 for each of the standard sets, respectively. Also, it was found that ATR spectra can be obtained from bacteria grown on blood agar and still have good differentiating capabilities for Gram stain. In addition, ATR spectra provide significantly better differentiating spectral information and predicting capabilities than specular reflectance spectra obtained in situ. Finally, the numerical value obtained for the Gram stain can be used as a measure of Gram variability. Results indicate that the Gram positive values shift to Gram negative values as the bacterial cell wall deteriorates with time.