The spread‐plate and double agar layer (DAL) methods are common for the enumeration of bacteria and viral indicators (bacteriophages). However, they may become cumbersome in large matrix experiments or when the titer of the organism varies by several orders of magnitude. A bacterial spot‐titer assay has been available for decades but has not been adapted to bacteriophages and has rarely been applied to the analysis of environmental samples. In this study, a spot‐titer culture‐based method was investigated for bacteria and bacteriophages. The method involves spot‐plating replicate 10‐µl volumes of several sample dilutions on a single plate, incubating, and counting colonies or plaques. Parallel assays of laboratory cultures and environmentally isolated organisms show that the spot‐titer method is equally straightforward and statistically comparable to the spread‐plate and DAL methods (R2 = 0.989 for laboratory strains and R2 = 0.972 for environmental samples), while more cost‐ and labor‐efficient. PRACTICAL APPLICATIONS The spread‐plate and double agar layer (DAL) methods currently used for enumeration of bacteria and viral indicators, may become labor‐ and resource‐intensive (culture media, plates, technician time and incubator space) in large matrix experiments, which are often needed in the laboratory to evaluate environmental conditions. The spot‐titer method has several advantages over the spread‐plate and DAL methods: (1) it requires less time to dispense spots than to spread the microbe; (2) it uses fewer materials (15–20% of the laboratory supplies as the traditional methods); (3) it requires less effort; and (4) since the sample is distributed in distinct spots, colony/plaque counting is faster and less labor intensive. The spot‐titer method was found to economize resources without sacrificing accuracy or precision, and is a practical method for routine use in large matrix experiments (e.g., survival or disinfection studies) and enumeration of high‐titer environmental samples.
Contaminated environmental surfaces are important reservoirs in the transmission of many human pathogens. Although several options exist for disinfecting contaminated environmental surfaces, few are compatible with use on both hard smooth non-porous (hard) and soft porous surfaces (soft) while still offering significant disinfection of the contaminating organisms. This study evaluated the efficacy of mist application of a stabilized chlorine dioxide and quaternary ammonium compound-based disinfectant (Cryocide20) for inactivation of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) on various environmental surfaces. MRSA and VRE were applied to hard and soft surfaces (glass, steel, tile, carpet, and cotton fabric), allowed to dry, and exposed to a uniform mist application of the disinfectant solution. After 1 hr of contact time, the residual disinfectant was neutralized, and the bacteria were recovered and enumerated on brain heart infusion (BHI) agar. Reduction of both test bacteria was observed on most of the hard and soft surfaces tested. Log(10) reduction of the organisms tended to be higher on steel, tile, and carpet than glass or cotton. Overall, these results suggest that mist application of Cryocide20 disinfectant may be an effective option for reduction of low levels of infectious bacterial pathogens from contaminated environmental surfaces.
The start-up of the Clark County Water Reclamation District (District) Moapa Valley Water Resource Center (MVWRC) presented several operational challenges. The influent flow and organic load was significantly less than the design minimum as the project schedule occurred during an unexpected economic turndown -that continues on today. In order to accelerate startup, the basins were seeded with thickened waste activated sludge from the District's Main Central Plant in Las Vegas. Instead of growing and stabilizing, the biomass essentially disappeared in a matter of weeks after seeding, resulting in a sharp decrease of the mixed liquor suspended solids concentration. Start-up troubleshooting indicated that a combination of solids settling, midge fly larvae invasion, uncontrolled solids wasting, and biofilm formation on tank walls caused the initial biomass instability. Inconsistent plant performance was also observed during start-up. Process modeling and field observations indicated that reduced organic load, excessive aeration, and biomass instability were the major causes of the inconsistent process performance. Process modifications were identified and implemented to address the start-up challenges experienced by District staff. Supplemental carbon was implemented on an emergency basis. The process configuration and aeration control were modified to minimize excessive aeration. The return activated sludge and mixed liquor internal recycle flows were adjusted to minimize solids settling in the structures. As a result of the start-up optimization effort, the MVWRC consistently exceeds both the design treatment goals and the mandated permit requirements.
The start-up of the Clark County Water Reclamation District (District) Moapa Valley Water Resources Center (MVWRC) presented several operational challenges. The influent flow and organic load was significantly less than the design minimum, and the project schedule occurred during the unexpected economic turndown. Based on the construction start-up schedule, there was minimal time for promoting biomass accumulation, so plant operations staff chose to import biomass from a larger plant to seed the MVWRC aeration basins. Instead of thriving and stabilizing, the biomass essentially disappeared in a matter of weeks after seeding. Start-up troubleshooting indicated that a combination of mixed liquor settling, proliferation of midge fly larvae, solids wasting during start-up, and thick biofilm formation on tank walls caused the initial biomass instability. The biomass instability, a reduced organic load, and excessive aeration during start-up lead to inconsistent plant performance. Process modeling confirmed the impact of reduced organic load and excessive aeration on the system. Process modifications were identified and implemented to address the start-up challenges experienced by District staff. The MVWRC is currently exceeding the design treatment goals and consistently meeting permit requirements.
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