Wild-type phage isolation. Wild-typ

Wild-type phage isolation. Wild-type phages were isolated using previously
described protocols (19, 23) with some variations. Wastewater samples were
collected from 14 different municipal wastewater treatment plants located
throughout Indiana. Samples were frozen at 20°C until processed. Thawed
samples (20 ml) were centrifuged (2,000  g for 10 min), and the supernatants
were filtered (0.2-m filters) and treated with chloroform (1:100). Filtered samples
were added to log-phase Salmonella enterica serovar Typhimurium in double-
strength tryptic soy broth (TSB; Difco, Sparks, MD) containing 50 g/ml
nalidixic acid and incubated at 37°C overnight with shaking. Overnight cultures
were centrifuged (1,000  g for 10 min), filtered (0.2-m filter), and treated with
chloroform (1:100). Serial 10-fold dilutions of the filtered samples (100 l) were
combined with 100 l of fresh log-phase S. Typhimurium, 67.5 l of 1 M CaCl2,
and 3 ml of nutrient agar (5%) overlay (containing 50 g/ml nalidixic acid). The
phage-overlay solution was then added to nutrient agar plates and incubated
overnight at 37°C. One isolate from each wastewater treatment plant (n  14)
was plaque purified three times, suspended in SM buffer (0.1 M NaCl, 1 mM
MgSO4, 0.2 M Tris [pH 7.5], 0.01% gelatin) containing 1% chloroform, and
stored at 80°C until further use.

ผลลัพธ์ (อังกฤษ) 2:

Wild-type phage isolation. Wild-Type phages were isolated using previously
described protocols (19, 23) with Some variations. Wastewater samples were
Collected Municipal Wastewater Treatment Plants from 14 different located
throughout Indiana. Samples were frozen at? 20 ° C until processed. Thawed
samples (20 ml) were Centrifuged (2,000? G for 10 min), and the supernatants
were filtered (0.2-? M filters) and treated with Chloroform (1: 100). Filtered samples
were Salmonella enterica serovar added to log-phase Double Typhimurium in
tryptic soy broth strength (TSB; Difco, Sparks, MD) containing 50? G / ml
nalidixic acid and incubated at 37 ° C with Shaking Overnight. Overnight cultures
were Centrifuged (1,000? G for 10 min), filtered (0.2-? M Filter), and treated with
Chloroform (1: 100). Serial dilutions of the 10-Fold filtered samples (100? L) were
combined with 100? L of S. Typhimurium Fresh log-phase, 67.5? L of 1 M CaCl2,
and 3 ml of nutrient agar (5%) overlay (containing. 50? g / ml nalidixic acid). The
phage-nutrient agar overlay Solution was then added to plates and incubated
at 37 ° C Overnight. One isolate from each Wastewater Treatment Plant (n? 14)
was plaque purified Three times, suspended in SM buffer (0.1 M NaCl, 1 mM
MgSO4, 0.2 M Tris [pH 7.5], .01% Gelatin) containing 1% Chloroform, and
stored. at? 80 ° C until further use.