Copper kills diarrhoeagenic bacteria countries. The existing community interventions to provide safe drinking-water to the people have many shortcomings, and studies have shown that point-of-use (PoU) household interventions con- tribute to 30-40% reduction in diarrhoeal diseases (10). Moreover, in countries such as India where only 28% of households have piped water (5), PoU interventions are a sustainable way to providing safe drinking-water. Storing water in copper and silver pots finds men- tion in ancient texts of Ayurveda for purification of water (11). Our previous study provided laboratory evidence of the antibacterial activity of copper pot in distilled water (12). We had also reported the benefit of using a copper-based device, contrived by us, which was as effective as the pot but at a fraction of the cost (12). Since distilled water is slightly acidic (pH 6.7±0.05) which might en- hance copper leaching, we have demonstrated the effect of copper pot in regular drinking-water (pH 7.83±0.4) against important bacterial pathogenic strains that cause diarrhoea. MATERIALS AND METHODS Bacterial strains V. cholerae O1 IDH 02474 (VC), S. flexneri type 2a IDH 02196 (SF), Salmonella enterica Typhi 500865 (SET), and enterotoxigenic E. coli (LT+ST) IDH 01254 (ETEC) were obtained from the National Ins- titute of Cholera and Enteric Diseases (NICED), Kolkata, India. S. Paratyphi A B/05 (SPT) was pro- cured from the St. Johns Medical College, Banga- lore, India and confirmed at NICED. Enteropatho- genic E. coli E 2347 (EPEC) was obtained from the Christian Medical College (CMC), Vellore, India. Preparation of bacterial cultures Cultures from the nutrient agar culture-stab were streaked onto selective media, including eosin methylene blue (EMB) agar medium (HIMEDIA, Mumbai, India) for E. coli species, xylose lysine dex- trose (XLD) medium (HIMEDIA) for Salmonella spe- cies, Henktoen enteric agar (HEA) medium (Difco, USA) for Shigella, and thiosulphate-citrate bile-salts sucrose (TCBS) medium (HIMEDIA) for V. cholerae, and were incubated at 37 0C for 16-18 hours in a bacteriological incubator (IN 18 DF, Servewell In- struments Private Limited, Bangalore, India). After incubation, a single colony was picked and inocu- lated into 2 mL of Luria Bertani broth (Difco) and incubated for 16-18 hours in a bacteriological incu- bator at 37 oC. This overnight culture was serially diluted in normal saline (NaCl, 0.85%) for inocula- tion in water. 18 Sudha VBP et al. Antibacterial activity of copper pot on drinking- water inoculated with enteric pathogens The experiment procedure followed was essen- tially as per Sudha et al. (12). Copper pots of 2-L capacity (test) purchased from local vendors were thoroughly cleaned and autoclaved each time be- fore use. Presterilized 1-L glass bottles (Schott Du- ran, Mainz, Germany) acted as controls. Water was collected from the tap (groundwater, pumped to the overhead tank) from the Microbiolo- gy Unit of FRLHT (Foundation for Revitalisation of Local Health Traditions), Bangalore and was auto- claved. The sterilized water was inoculated to ~500 colony-farming unit (CFU)/mL with serially- diluted overnight culture of the diarrhoeagenic bacteria. The same was enumerated by spread plate method on nutrient agar (HIMEDIA). Two litre of inoculated water was poured into copper pots (2x2L) and one litre into each of the two prest- erilized Schott Duran bottles. After incubation at room temperature (26±2 oC) for 16 hours, 100 μL of samples was withdrawn, after mixing, from each container and plated on nutrient agar for the enu- meration of bacteria. Resuscitation of sublethal- ly-damaged cells was monitored by enrichment method (13). Three mL of test or control water sample was mixed with an equal volume of double- strength peptone water (enrichment medium) and incubated for 24 hours at 37 oC. After incubation, the medium was observed for turbidity, and also a loopful of the enriched culture was streaked onto respective selective media as mentioned earlier and observed for growth after incubation for 24 hours at 37 oC. All experiments were conducted three times with duplicates maintained each time. Analysis of physical and chemical parameters of water Tests and controls of the inoculated water were assessed before and after incubation for physi- cochemical properties, including pH, turbidity, total dissolved solids (TDS), alkalinity, hardness, contents of chlorides and sulphates as per proto- cols of the Bureau of Indian Standards (14). The pH was measured using a pH meter (DI 707; Digisun Electronics, Hyderabad, India). Copper content was estimated using Spectroquant (Merck, Darmstadt, Germany), a commercially-available, ready-to-use kit, as described in Sudha et al. (12). RESULTS Antibacterial activity of copper pot on drink- ing-water inoculated with enteric pathogens VC, SF, ETEC, EPEC, SET, and SPT inoculated into water could not be recovered on the specific JHPN