In the laboratory, each sample bag was gently mixed by inverting and 100 ml poured into sterile plastic bottles. In addition, a 1:100 dilution of sample was prepared in sterile distilled water. E. coli in rinse samples were quantified using IDEXX™ QuantiTray®/ 2000 (Westbrook, ME, USA) most probable number (MPN) methodology and Colilert-18® media, according to manufacturer’s instructions. Positive controls (eluent water seeded with a lab-strain of E. coli) and lab blanks (eluent water only) were assayed each sampling day to validate test performance and to ensure lack of contamination in eluent water, respectively. Each sample was assigned into one of three categories based on estimated MPN of E. coli in 100 ml of hand rinse eluent: undetectable (<1 MPN), moderately contaminated (1e100 MPN), and highly contaminated (>100 MPN).
Quanti tray 2000
Manufactured by IDEXX
Sourced in United States
The Quanti-Tray/2000 is a lab equipment product manufactured by IDEXX. It is designed for the quantification of bacteria in water samples. The product utilizes a pre-defined substrate and specialized trays to enable automated enumeration of target microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Colilert 18, by IDEXX (3 mentions)
Enterolert, by IDEXX (2 mentions)
Colilert, by IDEXX (2 mentions)
Miseq instrument, by Illumina (1 mentions)
Quanti tray sealer plus, by IDEXX (1 mentions)
Escherichia coli, by American Type Culture Collection (1 mentions)
Tryptone, by BD (1 mentions)
Peptone, by BD (1 mentions)
Colilert 18 reagent, by IDEXX (1 mentions)
Noble agar, by BD (1 mentions)
Amicon ultra centrifugal filter unit, by Merck Group (1 mentions)
Yeast extract, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ethanolamine, by Merck Group (1 mentions)
16 mercaptohexadecanoic acid, by Merck Group (1 mentions)
Meat extract, by BD (1 mentions)
Whatman, by Cytiva (1 mentions)
Colilert reagent, by IDEXX (1 mentions)
Ics 2000 system, by Thermo Fisher Scientific (1 mentions)
20 protocols using quanti tray 2000
1
Quantification of E. coli in Hand Rinse Samples
2
Comprehensive Water Quality Analysis
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Duplicate samples were tested for E. coli, an indicator of fecal contamination, and total coliforms, via IDEXX Colilert Defined Substrate and IDEXX Quanti-Tray/2000 (IDEXX, Westbrook, MN, USA) at UVA-Wise and Virginia Tech (Standard Method 9223) [31 ]. Samples were also tested for nitrate (NO3−) and sulfate (SO42−) using a Hach DR850 portable colorimeter (Hach Company, Loveland, CO, USA). Additionally, samples were processed with 2% trace metal grade nitric acid by volume and then analyzed for metals including arsenic, cadmium, chromium, copper, iron, lead, manganese, and silver using a Thermo Electron iCAP-RQ ICP-MS at Virginia Tech (Standard Methods 3030D, 3125B) [31 ].
Considering the importance of identifying specific pathogenic organisms [32 (link)], source water from each household was filtered and concentrated using 0.2 μm concentrating pipettes (CP) for 1 L, and 0.05 μm CPs for 500 mL, with an InnovaPrep Concentrating Pipette Select, and then eluted in 0.075% Tween 20/25 mM Tris wet foam elution buffer (InnovaPrep LLC, Drexel, MO, USA). Elutions were shipped on dry ice from Virginia Tech to the University of Virginia for extraction and analysis using a custom-designed TaqMan array card, high throughput RT-qPCR assay, to detect 30+ viral, bacterial, protozoal, and helminthic pathogens [33 (link),34 (link),35 (link)].
Considering the importance of identifying specific pathogenic organisms [32 (link)], source water from each household was filtered and concentrated using 0.2 μm concentrating pipettes (CP) for 1 L, and 0.05 μm CPs for 500 mL, with an InnovaPrep Concentrating Pipette Select, and then eluted in 0.075% Tween 20/25 mM Tris wet foam elution buffer (InnovaPrep LLC, Drexel, MO, USA). Elutions were shipped on dry ice from Virginia Tech to the University of Virginia for extraction and analysis using a custom-designed TaqMan array card, high throughput RT-qPCR assay, to detect 30+ viral, bacterial, protozoal, and helminthic pathogens [33 (link),34 (link),35 (link)].
3
Enterolert-E vs. RPA-LFA for Enterococci
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The Enterolert-E test, the standard method (ISO 7899-1) for enterococci, was performed to compare the sensitivity with the RPA-LFA assay (elaborated in Section 2.9 ). The Enterolert-E test determines the presence/absence and numbers of enterococci based on the IDEXX Defined Substrate Technology (DST) nutrient indicator. This nutrient indicator produces fluorescence when metabolised by enterococci. The test was conducted according to the manufacturer’s instructions using the Quanti-Tray/2000 (Enterolert E, IDEXX, Rydalmere, New South Wales, Australia). The different dilutions of E. faecalis were prepared and artificially spiked into enterococci negative samples (tap water, saline, and wastewater). The inoculated samples were sealed in plates and incubated at 41 °C for 18–24 h. The plates were observed after 24 h under a UV illuminator. Blue illuminated wells were counted as positive for E. faecalis and the most probable number (MPN) calculated following the manufacturer’s instructions. Although MPN is calculated using the Enterolert-E test, selective plate isolations were used throughout the experiments to count the number of bacteria. For simplification, the bacterial counts obtained by all methods described here are expressed as the number of organisms/100 mL.
4
Comprehensive Environmental Microbiome Analysis
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Dissolved inorganic nutrient analysis for ammonium, orthophosphate, and nitrate were done by colorimetric analysis in microplates, as described previously (Ringuet et al., 2011 (link)). E. coli and Enterococci were enumerated using Colilert and Enterolert in the Quanti-Tray/2000 format following manufacturer recommendations (IDEXX, Westbrook, ME). Microbial community DNA for metagenomic analysis was recovered from the Sterivex cartridges as described previously (Amaral-Zettler et al., 2008 (link)) and assessed for molecular weight by agarose gel electrophoresis. These crude extracts were subsequently purified by passage through a OneStep PCR Inhibitor Removal column (Zymo, D6030) and purity was assessed by UV-spectra. Metagenomic analysis followed protocols outlined in the Earth Microbiome Project (Caporaso et al., 2011 (link)). Briefly, the V4 region of the 16S rRNA gene was amplified to generate an amplicon library. This library was multiplexed using Illumina designed indices, pooled with equal amounts, and sequenced on an Illumina MiSeq instrument as described by Caporaso et al. (2012) (link).
5
Assessing Water Point Suitability for SODIS
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Water points were mapped (GPS coordinates) and assessed for turbidity and microbiological quality to determine their suitability for SODIS treatment and the need, if any, for prefiltration of water. A community member assigned by village leadership helped in the identification of all water points used in the village. The GPS of each water point was taken in August 2017 (dry season) and December 2017 (rainy season). Specific data on each water point was collected using a standard questionnaire in KoBo Collect (https://www.kobotoolbox.org ).
Water quality tests were conducted in December 2017 to determine the turbidity (Turbidity meter‐HACH 2100Q) and microbiological quality based on most probable number (MPN) of coliforms and Escherichia coli (Colilert test:https://www.idexx.com/en/water/water‐productsservices/colilert/ ). Samples were taken directly from each water source in a sterilized container and placed in a cool box at <50 °C. Samples were delivered to the University of Malawi–Polytechnic laboratory within 3 h of sampling and processed immediately using the Colilert‐18 (IDEXX, UK) and Quantitray 2000 (IDEXX, UK) systems. Following 18 h of incubation, samples were read for MPN.
Water quality tests were conducted in December 2017 to determine the turbidity (Turbidity meter‐HACH 2100Q) and microbiological quality based on most probable number (MPN) of coliforms and Escherichia coli (Colilert test:
6
Quantification of E. coli in Environmental Samples
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E. coli was chosen as the fecal indicator bacteria because of the simple laboratory methods used for identification and quantification and their widespread use as a measure of fecal contamination [21 –24 (link)]. The concentration of E. coli in environmental samples can be measured using membrane filtration and m-ColiBlue24 ® broth media (Hach Company, Loveland CO) or Chromocolt ® Coliform Agar (EMD MilliporeSigma, Burlington, MA), or IDEXX-Colilert-24® and the Quanti-Tray/2000 (IDEXX Laboratories, Westbrook, ME) [25 –27 ]. Public or shared toilet surface swabs, raw produce, street food, and soil samples undergo a processing step prior to analysis for E. coli using protocols developed for the formative study [12 ]. Liquid samples do not require a processing step. Other internationally approved methods for quantifying E. coli can be substituted, but that may affect the sensitivity and reliability of the data. Two to three dilutions are analyzed for each sample in order to get a reliable estimate of concentration. To calculate the concentration of E. coli in a given sample, a selection step is used to choose dilutions for calculation (averaging) and identify any samples that present conflicting results between dilutions. The dilution protocol and process for calculating concentrations of E. coli are described in S1 Appendix .
7
Quantifying E. coli in Water Samples
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Water samples were analyzed for E. coli using the IDEXX Colilert-18 and Quanti-Tray 2000 method (IDEXX Laboratories, Westbrook, Maine), a defined substrate technology (Edberg et al., 1991 (link)). Generally, 100 ml of water was analyzed; excessively turbid samples were diluted as needed before analysis. Results are calculated as most probable number (MPN)/100 mL (Byappanahalli and Nevers, 2019 (link)).
8
Enumeration of Waterborne Coliforms and Enterococci
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Water samples (100 mL) were either processed directly or diluted when necessary into IDEXX (Westport, ME, United States) Colilert or Enterolert substrate for enumeration of total coliforms/E. coli and enterococci, respectively, as recommended by the manufacturer. In brief, the mixtures were then transferred to an IDEXX Quanti-Tray/2000, sealed using the IDEXX Quanti-Tray Sealer PLUS, and then, respectively, incubated at 35 and 41°C for 24 h as recommended by the manufacturer. Positive control organisms, consisting of Klebsiella pneumoniae subsp. pneumoniae (ATCC [American Type Culture Collection] 13883) and Escherichia coli (ATCC 25922) for Colilert and E. faecalis (ATCC 29212) for Enterolert, were utilized to confirm the effectiveness of each new substrate lot. After the 24-h incubation period, the Colilert and Enterolert trays were viewed under a 365-nm UV light, with blue fluorescing wells marked positive for E. coli and enterococci. In Colilert trays, yellow wells under normal lighting were marked positive for total coliforms. The number of positive wells were converted to most probable number (MPN) using the IDEXX result interpretation table and the dilution factor.
9
Quantitative Analysis of Fecal Indicator Bacteria
10
Comprehensive Monitoring of Tubewell Water Quality
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