3 Results

3.1 QCL-GAP spectra of microorganisms

The vibrational response obtained from the microorganisms using the QCL-GAP was used to determine the functional groups used for characterization. Because of the spectral similarities among bacterial species, it is difficult to discriminate using spectrum profile differences associated with sample homogeneity at the substrate surface. Sample distribution in the deposition technique used and the uniformity of the bacteria on the surface after the sample drying process also affect the discrimination of bacterial species. These factors can impact the band characterization and the discrimination processes on bacteria mixtures. The optical arrangement in the QCL-GAP creates an ellipse that covers a bigger surface area. This setup makes the beam size significantly larger than a typical IR system. Analysis of the spectral profile of the microorganisms under study shows characteristic signatures that allow identification and discrimination between the species.

Variation in the sample homogeneity during the deposition in the substrate is expected during spectral acquisition. However, the QCL-GAP ellipse effect can overcome those variations by covering more areas to enhance the signals during the spectrum acquisition. This effect played a role during the spectrum acquisition, providing more information that led to acceptable discrimination of the bacteria on pure and mixture of the bacteria.

The spectral measurements for the substrates (references or blanks) were measured at multiple regions of the deposited sample in the substrate. Before measuring the spectra of the neat bacteria and mixtures of bacteria, the spectrum of the substrate without the analyte is acquired as a reference. Then, the substrate containing the bacteria was collected. The instrument allows background subtraction without the need for additional processing. The performance was also verified before each run by collecting a blank spectrum using a gold (Au) substrate.

Band assignments in the fingerprint region for the bacteria sample’s present spectra variations due to the substrate’s reflectivity. In this experiment, the SS substrates used had a high reflectivity that allowed acquiring as many signals as possible. It is common knowledge that higher porosity of the substrate will reduce the signal to noise because it will absorb the light, resulting in fewer signals. That effect can lead to losing important information, impacting the resolution, and complicating the discrimination process.

Representative spectra for the Se and Ml mixture and neatSe and Ml spectra are shown in Figure 2. Although differences are observed in the mixture profile compared with the neat bacteria spectra, some bands have similarities. The same effect was observed for the other mixtures, such as Sa and Ml , shown in Figure 3, and Sa and Se , shown in Figure 4.