# Photocatalytic Inactivation as a Method of Elimination of _E. coli_ from Drinking Water
## 5 Quantitative Estimation of _Escherichia coli_  
One of the most common methods for estimating _E. coli_ is to use Ethidium monoazide bromide coupled with the quantitative polymerase chain reaction (EMA-qPCR) analysis. The method is used to validate other methods of _E. coli_ estimation. The pre-treatment carried out on _E. coli_ before carrying out the quantitative polymerase chain reaction involves the use of Ethidium monoazide bromide. This analysis works based on the membrane integrity of the bacteria. EMA binds to the DNA of the bacteria with damaged cell membrane and inhibits the bound DNA amplification when subjected to polymerase chain reaction. The amplification of the DNA would be done for bacteria cells with undamaged DNA [126]. The use of aerosol generator to estimate the population of _E. coli_ has also been reported. The suspension of _E. coli_ cells was aerosolized to less than 5 um to give bioaerosol containing bacteria. This was followed by a plate counting step and incubation of nutrient agar at a temperature that is above room temperature for a day [107]. In some cases, the incubation is assisted with rotary shaker or centrifuge at a particular rotation per minute before termination of the incubation after some minutes [109]. The counting on each nutrient agar plate is expressed in colony-forming units (cfu) [107]. Transcriptome analysis is another method of estimating the amount of _E. coli_. This method requires the isolation of RNA of the bacteria before the concentration and purity of the bacteria cells \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{1}{c}{**Photocatalyst**} & **Fabrication Method** & **Performance** & **Light Used** & **Reactive Species** & **Ref.** \\ \hline \multirow{4}{*}{conjugated polymer/ CuO nanoparticles} & \multirow{4}{*}{-} & Better bacteria & \multirow{4}{*}{UV-vis light was introduced to the polymer} & \multirow{4}{*}{UV-vis light was introduced to the polymer} & \multirow{4}{*}{UV-vis light was \(h^{*}\), \(\text{O}_{2}\) – and \(\text{H}_{2}\text{O}_{2}\)} & [117] \\  & & & Inactivation of 97\% & & \\ \cline{1-1}  & & _E. coli_ in 1 h & & & \\ \cline{1-1}  & & 96\% E. coli present in & & & \\ \cline{1-1}  & & 50 cm\({}^{3}\) 10\({}^{5}\) colony & & & \\ \cline{1-1}  & & - & forming units ml\({}^{-1}\) & & \(\text{`HO}_{2}\), \(\text{`OH}_{2}\), \(\text{`O}_{2}\)– & \\ \cline{1-1}  & & & solution were killed & & \\ \cline{1-1}  & & & in 50 min & & \\ \cline{1-1}  & & 99.997\% bacterial & & & \\ \cline{1-1}  & & process & inactivation & Visible light & \(\text{`OH}\) & [71] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._would be estimated by a spectrophotometer. The process is immediately followed by the use of bioanalyzer to determine the integrity of the RNA [56]. The use of fluorescence detector with 4\({}^{\prime}\)-6-diamidino-2-phenylindole has also been reported [122]. The working principle of this technique relies on the fact that 4\({}^{\prime}\)-6-diamidino-2-phenylindole stains the DNA of the bacteria with a damaged cell membrane. Since reactive oxygen species will damage the cell membrane of _E. coli_ during photocatalysis, in order to achieve accurate results, the sample containing the bacteria was rinsed several times with saline-phosphate buffer. The mixture of the stain and the treated sample was then excited and quantified via the fluorescence detector [122]. [PERSON] et al. [111] described the disc diffusion method as an alternative method of quantifying _E. coli_ inactivated by photocatalysis. The prepared sample was placed in a treated plate, and the plate was then exposed to visible light for less than 1 h and afterward inoculated for 10 h at 37 \({}^{\circ}\)C. An agar well diffusion method was also reported by [PERSON] et al. [127]. In this method, four small wells (diameter 0.8 mm) were dug and the stock solution of the photocatalyst was placed into the dug well, followed by the incubation above room temperature for one day while methanol was used as control [128].