sensor signals, 9 standard gas vectors were built in the space of a 10 sensor dimension. Then volatiles samples were measured and the information obtained was compared with the standard gas vectors. Finally, the data were described in terms of the standard gas categories (odour indices in standard gas categories). The calculation method for the indices was as shown in Figure 1b and as described in the Methods section mentioned below. The odour indices were quickly calculated by projecting the vectors against the standard gases categories. Moreover, the FF-2A was installed with a trap tube for concentrating smells and removing water vapour, which can affect the measurement value (Figure 1a) [22]. This system can maintain constant sample humidity and thereby, increase the reproducibility of the measurements. Depending on the aim of the analysis, use of this trap tube is optional. In this study, we challenged FF-2A with early detection of yeast and confirmed whether the combination method, absolute-value intensity and metaphor specification, is useful for detection and discrimination of microorganisms. To determine the lowest yeast concentration detectable, we measured the volatiles from samples obtained from 102 to 107 cfu/ml in the Glucose-Yeast-Peptone (GYP) media. In addition, to investigate the advantage of combining intensity and specification, we tested 2 other methods, i.e. total odour index (using smell intensity only) and similarity against other samples (using smell specification only). Materials and Methods Microorganisms and Culture Conditions Saccharomyces sp. (yeast) and Lactococcus lactis SNW-1 (lactic acid bacteria) were provided by Sanwa Norin Co. Ltd. These microorganisms were cultured in GYP media (1.0% glucose, 0.5% yeast extract, 0.5% peptone, 0.01% MgSO4, 0.0005% MnSO4, 0.0005% FeSO4, and 0.002% NaCl). Both microorganisms were cultured to 1.0 McFarland and then diluted to the indicated concentrations with the media. Measurements of Standard Gases and Volatile Samples The electronic nose was calibrated using the 9 standard gases as described, following which 2 ml of the samples (consisting of microorganisms and medium) were collected in 2-liter PET bags filled with dry nitrogen. The bags were allowed to equilibrate for 1 h at 25uC. The headspace volatiles were collected and diluted with dry nitrogen in new 2-liter PET bags. These diluted samples were introduced into the trap tube for 60 s and then exposed to the array with pure nitrogen gas. All the samples were measured four times and the final three measurements were used for analysis. Approximately 90 min were required to obtain the first data reading. Calculations The virtual concentration for each standard-gas axis was calculated by projecting the vector obtained to the axis (Figure 1b). The odour index for each category was calculated using equation (1). Odour index in standard gas category~10:log10ðCs=CtÞ ð Þ 1 Cs represented the virtual concentration compared to the standard-gas axis and Ct was used to describe the threshold concentration (the lowest detectable concentration) of the standard gas by the human nose. Real-Value & Metaphor Smell The total odour index, or the smell intensity as a whole, was calculated from the summation of the 9 standard gas intensities as in equation (2). Total odour index~10:log10 S ðCsi=CtiÞ ði~1{9Þ 2 ð Þ The similarity was calculated using the angles between the sample vectors. For this calculation we used the following criteria; h = 0u, similarity 100%; h.20u, similarity 0%. Results Analyses with Absolute-Value Intensity and Metaphor Expression Using the combination method, we showed the odour indices of the yeast volatiles in terms of the 9 standard gases categories (Figure 2). In the control media, organic acid was the highest category, and hydrogen sulphide and ammonia constituted the lower categories (odour index of the GYP media: organic acid, 23.5; hydrogen sulphide, 2.87; ammonia, 0.00). Over 102 cfu/ml, the indices obtained for the lower categories, hydrogen sulphide and ammonia, increased with concentration (Figure 2a). Over 104 cfu/ml, the indices for all the categories were higher than those obtained using the control media. These data suggested that Figure 2. Odour index changes generated by yeast volatiles determined by 9 standard gases categories. The graph (a) and radar chart (b) depict the values calculated by the odour index expressed as mean6standard deviation (n = 3). GYP indicates the GYP control medium. Lact 107 indicates the volatile sample from 107 cfu/ml of lactic acid bacterium in the GYP medium (bacterium control). doi:10.1371/journal.pone.0007939.g002 PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e7939