Evaluation of different indicator microorganism enumeration protocols for water quality monitoring
Type of DegreeMaster's Thesis
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Water quality, such as drinking water quality, irrigation water quality, and recreational water quality, is important for public health. Escherichia coli, coliform and Enterobacteriaceae have been used as indicator organisms to monitor the potential contamination of water. Previous studies indicate that bacterial concentrations may be significantly different when sampled at different times of the day. There are also studies showing that sediments contain higher levels of indicator microorganisms than the surface water. In recent years, in addition to E. coli and coliform, Enterococcus has been proposed to be used as an indicator organism for water quality due to its ability to survive in salt water. Although the literature provides important information, no parallel comparison among those enumeration methods have been conducted. The purposes of this study are: 1) to compare the efficiency of three enumeration methods for E. coli, including the mTEC membrane filter/USEPA method 1603, Coliscan® Easygel agar plates, and 3MTM PetrifilmTM method for field sampling; 2) to better understand the impact of the sampling times (morning vs. afternoon and months) and sample types (surface water vs. sediment) on water quality monitoring results; 3) to compare the efficacy of four different Enterococci enumeration protocols, including the mEI membrane filter/USEPA method 1600, the Enterolert® method, the Easygel cardsTM method and the USEPA qPCR method 1611, for freshwater monitoring. Our results show that there were no differences among the three E.coli enumeration methods (P > 0.05). Therefore, the Coliscan® Easygel agar plates method (used by the Alabama Water Watch) was used to evaluate the impact of sampling times and sample types on the enumeration of E. coli. Field sampling results show that both the sampling times and sample types may impact the enumeration results (P < 0.05), regardless of the indicator microorganisms used. When samples were collected in the afternoon, the surface water samples contained more indicator microorganisms than samples collected in the morning. Sediments contained more indicator microorganisms than the surface water (P < 0.05) and impacted the surface water monitoring results. The comparison of four Enterococci enumeration protocols show that while the Easygel cardsTM method has the lowest price ($1 per sample), the USEPA qPCR method 1611 ranks the highest among all tested methods based on the shorter processing time needed (~ 4 hours) and the widest detection range (2.47-8.47 log CFU/mL for surface water and 2.47-8.47 log CFU/g for sediment). Because of this, different DNA extraction methods were tested and compared to prepare samples for the qPCR protocol. Results show that, for surface water samples, the PrepMan® boiling procedure can substitute for the DNA extraction procedure used by the USEPA qPCR method 1611, however, for sediment samples, the PowerSoil® DNA Isolation Kit cannot be replaced by the PrepMan® boiling procedure. The results also show that the USEPA qPCR method 1611 is an efficient method for enumerating Enterococcus both in surface water and sediment.