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The coliphages were isolated from wastewater effluent of the Lehtoniemi municipal wastewater treatment plant (WWTP) located in Kuopio, Finland on 1st November, when the maximum outdoor temperature was 4.6 °C. The average influent flow to the WWTP was ca.18,700 m3/d in 2014. The wastewater samples (1,000 mL) were collected as two parallel subsamples and transported to the laboratory within 2 hours. Two host strains were used in order to isolate different coliphages. One of them was Escherichia coli ATCC 13706, which is widely used for detection of somatic coliphages and the other was E. coli ATCC 15597, used for detecting F-specific RNA coliphages (Hurst et al. 2007). The hosts were used for the isolation of coliphages from both subsamples according to the protocol described by ISO (1998), using the dilutions of 100, 10−1, and 10−2 of wastewater and host strains in the Log-phase. The host bacteria were taken from stock cultures with a sterile loop and inoculated in phage THG broth, consisting of tryptose 10 g, yeast extract 5 g, glucose 2 g, NaCl 5 g, and MgSO4·7H2O 0.25 g added to 1,000 mL of deionized water, and incubated at 37 °C for 24 ± 3 h. After incubation, 3.0 mL of suspension was re-inoculated into 100 mL phage THG broth and incubated at 37 °C for 24 ± 3 h. This overnight culture was rejuvenated in a shaker incubator (Edison, NJ, USA) at 37 °C for 2 h. The coliphages were cultivated by a double-layer technique as described by Adams (1959) using phage THG media with 12 g agar in hard agar and with 6 g agar in semi-solid agar in 1 L of deionized water. The method was modified so that 0.1 mL of 2,3,5-triphenyltetrazolium chloride (TTC) solution was added to 1.0 mL of semi-solid agar. This solution was prepared freshly by adding 0.04 g TTC to 5.0 mL of ethanol (Rajala-Mustonen & Heinonen-Tanski 1994). Different plaques were picked up from the plate depending on their size and morphology of lysogenic and lytic zones (Tan et al. 2008) and inoculated into fresh host solutions. The solution was incubated for 4–5 h at 37 °C and then left to stand for 24 h at 4 °C. The suspension was centrifuged at 3,250 × g for 15 min to separate bacterial cells and cell debris (in pellet) and coliphages (in supernatant) (Rajala-Mustonen & Heinonen-Tanski 1994). Purity of the coliphage suspension was verified by recultivating the solution two to three times by a double layer technique. The density of coliphages was titered by a double layer technique according to ISO (1998). In total, 17 different coliphages were isolated and purified (Table 1). The concentration of coliphages in these stock solutions was approximately 109 PFU/mL. MS2 (strain ATCC 15597-B1), an F-specific RNA coliphage, was similarly rejuvenated and used in the disinfection experiments.

Table 1

Diameter of lytic and lysogenic zones (mm, mean ± SD, n = 10) of plaques for 17 tested coliphages and MS2 coliphage using hosts E. coli ATCC 13706 and ATCC 15597

Coliphage ATCC number ofHost ATCC 13706Host ATCC 15597
isolation hostLytic zoneLysogenic zoneLytic zoneLysogenic zone
15597 2.09 ± 0.18 No zone 2.40 ± 0.48 1.63 ± 0.49 
15597 No plaques No plaques 1.42 ± 0.16 No zone 
15597 No plaques No plaques 2.87 ± 0.43 1.31 ± 0.51 
15597 0.74 ± 0.14 1.17 ± 0.26 1.05 ± 0.37 1.17 ± 0.26 
15597 2.78 ± 0.04 1.33 ± 0.06 2.72 ± 0.53 1.32 ± 0.32 
15597 2.23 ± 0.10 2.55 ± 0.16 2.12 ± 0.47 2.64 ± 0.67 
13706 1.85 ± 0.55 1.05 ± 0.30 2.27 ± 0.16 2.03 ± 0.20 
13706 1.76 ± 0.28 No zone 3.64 ± 0.26 1.97 ± 0.56 
13706 4.14 ± 0.30 1.99 ± 0.57 4.43 ± 0.15 2.17 ± 0.19 
10 13706 1.34 ± 0.41 No zone 0.80 ± 0.07 0.88 ± 0.11 
11 13706 1.80 ± 0.38 No zone 3.65 ± 0.42 3.70 ± 0.60 
12 15597 1.73 ± 0.13 No zone 3.56 ± 0.28 3.00 ± 0.36 
13 13706 4.69 ± 0.35 1.40 ± 0.16 4.71 ± 0.11 1.66 ± 0.20 
14 15597 Not tested Not tested 0.86 ± 0.40 No zone 
15 15597 No plaques No plaques 2.04 ± 0.32 No zone 
16 15597 3.60 ± 0.12 No zone 3.67 ± 1.31 No zone 
17 13706 3.56 ± 0.49 1.63 ± 0.31 3.34 ± 0.31 3.33 ± 0.65 
MS2 15597 No plaques No plaques 2.49 ± 0.29 1.21 ± 0.34 
Coliphage ATCC number ofHost ATCC 13706Host ATCC 15597
isolation hostLytic zoneLysogenic zoneLytic zoneLysogenic zone
15597 2.09 ± 0.18 No zone 2.40 ± 0.48 1.63 ± 0.49 
15597 No plaques No plaques 1.42 ± 0.16 No zone 
15597 No plaques No plaques 2.87 ± 0.43 1.31 ± 0.51 
15597 0.74 ± 0.14 1.17 ± 0.26 1.05 ± 0.37 1.17 ± 0.26 
15597 2.78 ± 0.04 1.33 ± 0.06 2.72 ± 0.53 1.32 ± 0.32 
15597 2.23 ± 0.10 2.55 ± 0.16 2.12 ± 0.47 2.64 ± 0.67 
13706 1.85 ± 0.55 1.05 ± 0.30 2.27 ± 0.16 2.03 ± 0.20 
13706 1.76 ± 0.28 No zone 3.64 ± 0.26 1.97 ± 0.56 
13706 4.14 ± 0.30 1.99 ± 0.57 4.43 ± 0.15 2.17 ± 0.19 
10 13706 1.34 ± 0.41 No zone 0.80 ± 0.07 0.88 ± 0.11 
11 13706 1.80 ± 0.38 No zone 3.65 ± 0.42 3.70 ± 0.60 
12 15597 1.73 ± 0.13 No zone 3.56 ± 0.28 3.00 ± 0.36 
13 13706 4.69 ± 0.35 1.40 ± 0.16 4.71 ± 0.11 1.66 ± 0.20 
14 15597 Not tested Not tested 0.86 ± 0.40 No zone 
15 15597 No plaques No plaques 2.04 ± 0.32 No zone 
16 15597 3.60 ± 0.12 No zone 3.67 ± 1.31 No zone 
17 13706 3.56 ± 0.49 1.63 ± 0.31 3.34 ± 0.31 3.33 ± 0.65 
MS2 15597 No plaques No plaques 2.49 ± 0.29 1.21 ± 0.34 

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