# A Lab-Made E-Nose-MOS Device for Assessing the Bacterial Growth in a Solid Culture Medium
## 2 Materials and Methods
### HS-SPME-GC-MS Evaluation of VOCs Emitted by Each Bacteria  
The profiles of VOCs emitted by the four bacteria studied, which were separately grown, were established by headspace solid-phase microextraction (HS-SPME) and GC-MS. The volatile fraction was chromatographically evaluated by analyzing the HS of glass vials (50 mL, Duran, Germany) containing 10 mL of BHI agar, previously inoculated with 100 \(\upmu\)L of a diluted overnight culture (10\({}^{-4}\) dilution for _E. faecalis_ and 10\({}^{-5}\) for the others bacterial strains). The vials were sealed with polytetrafluoroethylene/silicone screw caps and incubated at 37 \({}^{\circ}\)C for 24 h. The HS of the non-inoculated BHI culture medium was also analyzed and used as negative control. A Shimadzu GC-2010 Plus chromatograph with a mass spectrometer Shimadzu GC-MS-QP2010 SE detector was used. Each vial, inoculated with a pre-established amount of one of the four bacteria, was spiked with an accurate amount of 5 \(\upmu\)L of the internal standard (IS: \(\alpha\)-pinene, 98% from Sigma Aldrich, St. Louis, MO, USA) methanolic solution, with a concentration of 0.50225 mg/mL, being the volatiles adsorbed into an SPME fiber coated with divinylbenzene/carbonex/polydimethylsiloxane (DVB/CAR/PDMS 50/30 \(\upmu\)m, from Supelco, Bellefonte, PA, USA). The vials were conditioned for 5 min at 37 \({}^{\circ}\)C to allow the release of the VOCs. After this time period, the SPME fiber was exposed for 30 min, at 37 \({}^{\circ}\)C, allowing the adsorption of the volatile compounds present in the headspace. The volatile fraction of the solid medium not inoculated with any of the four bacteria was also studied following the same procedure and after being spiked with the same amount of IS. The procedure was repeated twice for each bacterium and not inoculated medium.  
As previously described [31], peaks' separation was accomplished on a TRB-5 MS (30 m \(\times\) 0.25 mm \(\times\) 0.25 \(\upmu\)m) column (Tekonkroma, Spain). The injector was set at 220 \({}^{\circ}\)C and the manual injections were made in splitless mode. The mobile phase consisted of helium (Praxair, Portugal) at a linear speed of 30 cm/s and a total flow of 24.4 mL/min. The oven gradient temperature was as follows: 40 \({}^{\circ}\)C/1 min; 2 \({}^{\circ}\)C/min until 220 \({}^{\circ}\)C (30 min). The ionization source was maintained at 250 \({}^{\circ}\)C with an ionization energy of 70 electronvolts (eV) and an ionization current of 0.1 kilovolts (kV). All mass spectra were acquired by electron ionization, being the spectra fragments identified by comparing with the mass spectra's databases of the NIST SRD-69 Library (National Institute of Standards and Technology, Gaithersburg, MD, USA) and with the free chemical structure/information databases of PubChem and ChemSpider. The minimum similarity for identification purposes was set equal to 85%. Furthermore, the peaks' identification was further checked using Kovat's retention indices. The areas of the chromatographic peaks were determined by integrating the re-constructed chromatogram from the full scan chromatogram using the ion base (m/z intensity 100%) for each compound. For semi-quantification purposes, the amounts of the identified volatiles were calculated by the ratio of each base ion peak area to the area of the internal standard base ion peak area, without considering the response factors, and converted to mass equivalents based on the internal standard concentration used.