Field Evaluation of Two Colorimetric Coliphage Detection Methods

APPLIED

AND

ENVIRONMENTAL MICROBIOLOGY, Mar. 1994, p. 826-830

Vol. 60, No. 3

0099-2240/94/$04.00 + 0 Copyright © 1994, American Society for Microbiology

Field Evaluation of Two Colorimetric Coliphage Detection Methods M. MARIAN IJZERMAN,I* JOSEPH 0. FALKINHAM 111,2 RAYMOND B. RENEAU, JR.,' AND CHARLES HAGEDORN' Department of Crop and Soil Environmental Sciences1 and Department of Biology, 2 Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received 27 September 1993/Accepted 5 January 1994

Two new methods for coliphage detection, a colorimetric agar-based (CAB) method and a liquid colorimetric presence-absence (LCPA) method, were compared to the coliphage method proposed by the American Public Health Association (APHA; Standard Methods for the Examination of Water and Wastewater, 18th ed., American Public Health Association, Washington, D.C., 1992). Both new methods are based on the induction of ,-galactosidase in Escherichia coli and the release of the enzyme through a lytic cell infection. The released enzyme then cleaves a chromogenic substrate which produces a colored reaction product. Ninety split water samples from four different sources were tested. A total of 52 samples were positive by the CAB method, 52 were positive by the LCPA method, and 53 were positive by the APHA method. Results indicated that (i) the CAB and LCPA methods were as sensitive in coliphage detection as the APHA method, (ii) both the CAB and LCPA methods were easier to read and interpret than the APHA method, and (iii) the CAB method detected more coliphages in a positive sample than the APHA method in two of the four types of water sources. Importantly, the rapid and simple LCPA method was as reliable and sensitive as either of the two agar-based methods in coliphage detection.

Currently, only one method has been proposed by the American Public Health Association (APHA) for the detection and enumeration of coliphages from ground and surface waters, the single-agar-layer plaque method (1). The APHA (1) method uses American Type Culture Collection strain Escherichia coli C (ATCC 13706) as the host because it lacks restriction and modification systems and is sensitive to a broad spectrum of coliphages in sewage (7). Further, the APHA method employs 2,3,5-triphenyltetrazolium chloride in the agar medium to aid in detecting plaques. As noninfected E. coli cells grow, the 2,3,5-triphenyltetrazolium chloride is reduced and colors the agar a pale pink, while plaques remain colorless. However, there are three main limitations associated with the APHA method for the detection of coliphages in water samples. The first limitation is the difficulty in accurately determining a coliphage count due to a lack of a significant color contrast on an agar plate (9). The second limitation is the inability to detect low numbers of coliphages (12). The APHA recommends its procedure for use only when there are greater than 5 PFU per 100 ml of sample (1). The third limitation is the appearance of plaque-like areas in the agar which, when counted, can lead to false-positive results (15). Two new coliphage detection methods, a colorimetric agarbased (CAB) method (9) and a liquid colorimetric presenceabsence (LCPA) method (8), have recently been developed to overcome the limitations imposed by the APHA method. Both new methods are based on the phage-induced lysis and release of induced 1-galactosidase from E. coli C. Once the enzyme is released, it hydrolyzes J3-galactosides coupled to chromogenic molecules, which results in the release of the chromogen and the formation of a unique color product that indicates the presence of coliphages in the sample. In laboratory studies, the CAB method proved to be superior in coliphage detection to the APHA method by being easier to read and interpret and by

detecting approximately twice the number of coliphage particles (9). The LCPA method also proved to be superior to the APHA method in laboratory studies by being more rapid, simpler to perform, and highly sensitive, and the results are easier to read and interpret (8). The purpose of this study was to perform a field evaluation comparing the CAB and LCPA methods with the APHA method by using water samples collected from a variety of sources. MATERIALS AND METHODS

Samples. Ninety water samples were collected from four types of sources within and around Blacksburg, Va., over the 2-month period from February to March 1993. The four sample sources were chosen because they provided a range of sample types with different levels of fecal contamination. Sample sources were as follows: (i) tap water collected from a private residence located at the end of the public water distribution line in the city of Blacksburg, Va., (ii) spring water collected from a private residence in Giles County, Va., (iii) creek water collected below the natural drainage of the dairy pastures at Virginia Polytechnic Institute and State University (VPI & SU), and (iv) wastewater collected from the sedimentation basin at the Blacksburg-VPI & SU Sanitation Authority Stroubles Creek wastewater treatment plant. The tap and wastewater samples were to serve as negative and positive controls, respectively, while the spring and creek water samples were presumed to vary between the two control sources. Sample volumes collected from the tap, spring, creek, and wastewater sources were 1.5 and 3.5 liters and 500 and 500 ml, respectively. Samples were collected in presterilized polypropylene or glass containers, transported to the laboratory, and analyzed within 2 h of collection. Sodium thiosulfate was added at a final concentration of 100 mg/liter in order to dechlorinate the tap water samples. Chemical and biological water analyses. Potassium (K), calcium (Ca), magnesium (Mg), manganese (Mn), iron (Fe), sodium (Na), zinc (Zn), and copper (Cu) were measured with

* Corresponding author. Mailing address: Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268. Phone: (513) 569-7387. Fax: (513) 569-7170.

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TABLE 1. Selected water quality parameters of the four experimental water sources Concn of substance (mg/liter) in solution

Water

source"

Tap Spring Creek Wastewater

pH

BOD51

K

Ca

Mg

Mn

Fe

Na

Zn

Cu

6.65 6.84 7.04 7.47

7.90 6.40 7.30 9.30

1.27 0.76 5.88 8.47

10.54 66.62 57.97 30.97

4.16 3.00 20.94 12.02

NDC 0.01 0.04 0.02

ND 0.53 0.82 0.08

5.81 1.50 21.50 32.40

0.63 0.02 0.02 0.06

0.15 0.01 ND 0.01

a Tap water was obtained from a private residence located at the end of the water distribution system in the city of Blacksburg, Va. Spring water was obtained from a private residence in Giles County, Va. Creek water was obtained below the dairy pastures at VPI & SU. The wastewater sample was collected from the sedimentation basin at the Blacksburg-VPI & SU Sanitation Authority Stroubles Creek wastewater treatment plant. b BOD5, 5-day biochemical oxygen demand. c ND, not detectable below analytical range: Mn,