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Volume 5, Number 3—June 1999


Chlorine Inactivation of Escherichia coli O157:H7

Eugene W. RiceComments to Author , Robert M. Clark, and Clifford H. Johnson
Author affiliations: U.S. Environmental Protection Agency, Cincinnati, Ohio, USA

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Chlorine inactivation of Escherichia coli O157:H7 and wild-type E. coli a

After exposure time of
Isolate Initial
noculum 30 sec 60 sec 120 sec Inactivation
rate (min-1) r2
E. coli O157:H7
N009-6-1 5.63 2.60 1.88 0.82 -2.96 0.82
N6001-8-10 5.78 2.52 1.44 0.72 -3.06 0.68
N6021-5-1 5.78 2.54 1.52 0.66 -3.06 0.54
N60049-26-1 5.68 2.35 1.40 0.54 -3.00 0.86
N6059-7-2 5.72 2.42 1.74 0.86 -3.02 0.72
N6104-5-9 5.62 2.40 1.69 0.72 -2.96 0.89
N6114-7-2 5.63 2.52 1.66 0.89 -2.96 0.82
Mean 5.69 2.48 1.62 0.74 -2.93 0.82
E. coli (wild type)
A 5.53 2.66 1.80 1.52 -2.51 0.61
B 5.79 2.60 1.48 0.81 -2.68 0.60
C 5.68 2.48 0.92 0.84 -2.61 0.61
D 5.52 2.34 0.95 0.39 -2.50 0.61
Mean 5.63 2.52 1.28 0.89 -2.93 0.71

aIn chlorine demand-free chlorinated (CDF) buffer, 5ºC, pH 7.0, 1.1 mg/L free chlorine, 1.2 mg/L total chlorine. Duplicate chlorine inactivation experiments were conducted in CDF buffer at pH 7.0. All experiments were conducted at 5ºC in a recirculating, refrigerated water bath. The chlorinated buffer was prepared by the addition of reagent-grade sodium hypochlorite (Fisher Scientific, Fair Lawn, NJ). Reaction vessels were continuously mixed (250 rpm) by using an overhead stirring apparatus equipped with sterile stainless steel paddles. Chlorine concentrations were determined by the N,N-dimethyl-p-phenylenediamine colorimetric method (9). Samples were removed from the reaction vessels at the desired exposure times, and the chlorine was immediately neutralized by the addition of 0.5 ml of 10% (wt/vol) sodium thiosulphate. Vessels containing CDF buffer without chlorine served as controls for determining unexposed concentrations of the bacteria. Initial levels and the number of survivors after chlorine exposure were determined by the membrane filtration procedure using mT7 agar incubated for 22 to 24 hours at 35ºC. This medium was chosen because of its ability to recover oxidant-stressed organisms (9). Levels of bacteria were determined by duplicate filtrations of appropriate dilutions for each exposure time. The log10-transformed data were used to determine the levels of inactivation for each isolate. The means for the inactivation data for the E. coli O157:H7 isolates and for the wild-type E. coli isolates at each exposure time were used to compare the inactivation rates between the pathogenic and the wild-type organisms. The following first order model was used to describe the inactivation rate: y = y1010-at, , where t = time in seconds, y = CFU/ml at any time t, y10 = CFU/ml at time zero, and a = the inactivation rate in min-1. The log transformation of this equation was used to calculate the inactivation rate. A regression analysis using least squares was conducted for experiments with each individual isolate and for the mean values for each of the two types of isolates (serotype O157 and wild-type) to determine the inactivation rates ("a" values).

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