# Detecting Multiple Mixed Bacteria Using Dual-Mode Hyperspectral Imaging and Deep Neural Networks
## 2 Materials and Methods
### Sample Preparation  
Four types of bacterial pathogen, including _Staphylococcus aureus_ (_S. aureus_), _Escherichia coliform_ (_E. coli_), _Pseudomonas aeruginosa_ (_P. aeruginosa_), and _Serratia marcescens_ (_S. marcescens_), were studied in this paper. These bacterial samples were provided by Taizhou Enze Medical Center, came from various clinical diagnosis samples including blood, sputum, urine, tissue fluid, and so on.  
The bacterial separation and cultivation processes are illustrated as follows. First, using the streak plate method, the originally mixed bacteria in the clinical samples grew and multiplied along the lines on the plate surface and formed dispersive individual colonies. A single colony should represent a bacterium in the original sample. These separated bacterial strains were then transplanted to a new culture dish (blood plates), and cultivated in incubators at 37 \({}^{\circ}\)C for about 48 h to produce purified colonies. During experiments, a portion of the purified bacteria was taken from the cultured dish and the bacterial samples were dissolved into sterile deionized (DI) water to prepare bacterial solutions for experimental measurement. The concentrations of the four pathogenic bacteria were about \(10^{9}\)/mL, measured by conventional cell counting method.  
After preparing the above single bacteria samples, 84 groups of mixed bacterial samples composed of different proportions of _S. aureus_, _E. coli_, _P. aeruginosa_, and _S. marcescens_ were also prepared. The total volume of the mixed bacteria samples in each group was maintained at 28 \(\upmu\)L. Table 1 shows the exact volume ratio of the four types of bacteria.  
\begin{table}
\begin{tabular}{c c c c c} \hline \multirow{2}{*}{**Sample**} & _E. coli_ & _S. marcescens_ & _P. aeruginosa_ & _S. aureus_ \\  & **(Ratio)** & **(Ratio)** & **(Ratio)** & **(Ratio)** \\ \hline
1–7 & 0.1 & 0.1 & 0.1, 0.2, \(\ldots\rightarrow\) 0.7 & 0.7, 0.6, \(\ldots\rightarrow\) 0.1 \\
8–13 & 0.1 & 0.2 & 0.1, 0.2, \(\ldots\rightarrow\) 0.6 & 0.6, 0.5, \(\ldots\rightarrow\) 0.1 \\
14–18 & 0.1 & 0.3 & 0.1, 0.2, \(\ldots\rightarrow\) 0.5 & 0.5, 0.4, \(\ldots\rightarrow\) 0.1 \\
19–22 & 0.1 & 0.4 & 0.1, 0.2, \(\ldots\rightarrow\) 0.4 & 0.4, 0.3, \(\ldots\rightarrow\) 0.1 \\
23–25 & 0.1 & 0.5 & 0.1, 0.2, 0.3 & 0.3, 0.2, 0.1 \\
26, 27 & 0.1 & 0.6 & 0.1, 0.2 & 0.2, 0.1 \\
28 & 0.1 & 0.7 & 0.1 & 0.1 \\
29–34 & 0.2 & 0.1 & 0.1, 0.2, \(\ldots\rightarrow\) 0.6 & 0.6, 0.5, \(\ldots\rightarrow\) 0.1 \\
35–39 & 0.2 & 0.2 & 0.1, 0.2, \(\ldots\rightarrow\) 0.5 & 0.5, 0.4, \(\ldots\rightarrow\) 0.1 \\
40–43 & 0.2 & 0.3 & 0.1, 0.2, \(\ldots\rightarrow\) 0.4 & 0.4, 0.3, \(\ldots\rightarrow\) 0.1 \\
44–46 & 0.2 & 0.4 & 0.1, 0.2, 0.3 & 0.3, 0.2, 0.1 \\
47, 48 & 0.2 & 0.5 & 0.1, 0.2 & 0.2, 0.1 \\
49 & 0.2 & 0.6 & 0.1 & 0.1 \\
50–54 & 0.3 & 0.1 & 0.1, 0.2, \(\ldots\rightarrow\) 0.5 & 0.5, 0.4, \(\ldots\rightarrow\) 0.1 \\
55–58 & 0.3 & 0.2 & 0.1, 0.2, \(\ldots\rightarrow\) 0.4 & 0.4, 0.3, \(\ldots\rightarrow\) 0.1 \\
59–61 & 0.3 & 0.3 & 0.1, 0.2, \(\ldots\rightarrow\) 0.3 & 0.3, 0.2, 0.1 \\
62, 63 & 0.3 & 0.4 & 0.1, 0.2 & 0.2, 0.1 \\
64 & 0.3 & 0.5 & 0.1 & 0.1 \\
65–68 & 0.4 & 0.1 & 0.1, 0.2, \(\ldots\rightarrow\) 0.4 & 0.4, 0.3, \(\ldots\rightarrow\) 0.1 \\
69–71 & 0.4 & 0.2 & 0.1, 0.2, 0.3 & 0.3, 0.2, 0.1 \\
72, 73 & 0.4 & 0.3 & 0.1, 0.2 & 0.2, 0.1 \\
74 & 0.4 & 0.4 & 0.1 & 0.1 \\
75–77 & 0.5 & 0.1 & 0.1, 0.2, 0.3 & 0.3, 0.2, 0.1 \\
78, 79 & 0.5 & 0.2 & 0.1, 0.2 & 0.2, 0.1 \\
80 & 0.5 & 0.3 & 0.1 & 0.1 \\
81, 82 & 0.6 & 0.1 & 0.1, 0.2 & 0.2, 0.1 \\
83 & 0.6 & 0.2 & 0.1 & 0.1 \\
84 & 0.7 & 0.1 & 0.1 & 0.1 \\ \hline \end{tabular}
\end{table}
Table 1: The composition of the four mixed bacteria samples.