Modification of Delayed-Incubation Procedure for Detection of

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1983, p /83/ $02.00/0 Copyright 1983, American Society for Microbiology Vol. 46, No. 4 Modification of Delayed-Incubation Procedure for Detection of Fecal Coliforms in Water MIN CHEN* AND PAUL J. HICKEY Center for Laboratories and Research, New York State Department of Health, Albany, New York Received 20 April 1983/Accepted 9 August 1983 Three holding media, including the vitamin-free Casitone holding medium (m- VFC) recommended by Standard Methods for the Examination of Water and Wastewater for use with the delayed-incubation membrane filter procedure, were compared for their ability to maintain viability of fecal coliforms. Each medium was tested according to the procedure described in the above reference with 60 to 80 pure cultures of fecal coliforms and a variety of natural water samples containing fecal coliforms. Fecal coliform recovery with m-st holding medium (containing ethanol, sulfanilamide, and Tris [ph 8.6]) was significantly greater than recovery with m-vfc (containing vitamin-free casein hydrolysate, sodium benzoate, sulfanilamide, and ethanol). Recovery with m-vfc, was, in turn, significantly greater than with NSB medium (containing nutrient broth, boric acid, and NaCl as major ingredients). Fecal coliform counts obtained with m-st by the delayed-incubation membrane filter procedure were higher than counts obtained by the standard immediate incubation. This result suggested that some of the sublethally injured fecal coliforms in natural water samples may have recovered during the incubation period. We propose that m-st be used in place of m-vfc for the delayed-incubation membrane filter procedure. Standard Methods for the Examination of Water and Wastewater (Standard Methods) (1) recommends that bacteriological examination of surface water samples be initiated within 1 h of the time of collection (or within 6 h if the sample is stored on ice). Difficulties in meeting these time requirements, particularly with samples taken in remote areas, led to the development of delayed-incubation test methods for field use (4, 6, 9). Each of these methods requires the use of a holding medium to stabilize bacteria trapped on the surface of a membrane filter while the filter is transported to the laboratory. In Great Britain a medium composed of 0.02% peptone water with 0.4% sodium benzoate was used (6) but was later found to be unsatisfactory (9). Taylor et al. (9) developed a vitamin-free Casitone holding medium (m-vfc) which is recommended in Standard Methods for use when the standard immediate-incubation membrane filter procedure (IMF) cannot be performed. Preliminary tests in our laboratory with surface water samples from six collection sites along Lake George in eastern New York State showed that counts obtained with the recommended delayed-incubation membrane filter procedure for fecal coliforms (DMF) were significantly lower and considerably more variable than those obtained by the standard IMF. This observation prompted our search for an alternative holding medium for use with the DMF. Several new holding media have been devised for bacterial proficiency test samples (3, 10). Toombs and Connor (10) have reported that NYSDH-1 holding medium is capable of maintaining 88% viability of an Escherichia coli strain for at least 2 days. Brodsky et al. (3) have described NSB, a medium containing nutrient broth, NaCl, and boric acid and capable of holding E. coli for up to 10 days. In this study we compared the effectiveness of DMF with three holding media: m-vfc, NSB, and a modified NYSDH-1 medium (m-st). The results provide the basis for a more effective DMF. 889 MATERIALS AND METHODS Preparation of holding media. m-vfc was prepared according to the method of Taylor et al. (9), except that vitamin-free enzymatic hydrolysate of casein was substituted for vitamin-free Casitone (Difco Laboratories, Detroit, Mich.), which was not available. NSB

2 890 CHEN AND HICKEY TABLE 1. Composition of m-vfc, NSB, and m-st holding media Manu- Amt in: Ingredient facturera m-vfc NSB m-st Vitamin-free casein ICN 0.2 g hydrolysate Sodium benzoate FS 0.4 g Sulfanilamide SC 0.5 g 1.5 g Ethanol (95%) US 10.0 ml 10.0 ml Potassium phos- JTB 3.0 g phate, dibasic Sodium phosphate, JTB g monobasic Tris FS g Boric acid FS g Sodium chloride FS g Nutrient broth DL g Distilled water 1 liter 1 liter 1 liter ph a DL, Difco Laboratories, Detroit, Mich.; FS, Fisher Scientific Co., Pittsburgh, Pa.; ICN, ICN Pharmaceuticals, Inc., Cleveland, Ohio; JTB, J. T. Baker Chemical Co., Phillipsburg, N.J.; SC, Sigma Chemical Co., St. Louis, Mo.; US, U.S. Industrial Chemicals Co., Louisville, Ky. was prepared according to the method of Brodsky et al. (3). m-st was NYSDH-1 (10) plus 0.3% Tris, which was added to improve buffering at ph 8.6. The composition of each of these holding media is shown in Table 1. Sources and verification of cultures. Some cultures were obtained from the American Type Culture Collection (ATCC strains 26, 128, 11775, and 25922). Others were obtained by isolating fecal-coliform-like colonies from various natural water samples on m-fc agar medium. The isolates were verified as fecal coliforms by using lauryl tryptose broth and EC medium (2). 0 Procedure. Each fecal coliform isolate was grown overnight at 35 C in a test tube containing 10 ml of 0.8% nutrient broth (Difco). To estimate bacterial densities, the turbidity of each culture was measured at 660 nm in a Bausch & Lomb Spectronic 70 spectrophotometer with a 1-cm light path. Each culture was then diluted in phosphate-buffered dilution water (1) and divided into 16 portions of identical volume, each with an estimated 50 to 75 organisms. Each portion was filtered through a separate, sterile Millipore membrane filter (0.45,um). Four filters were incubated immediately at C for h on m-fc agar medium. The remaining 12 filters were divided into three groups of 4 each for use with the three holding media. Each of these 12 filters was placed in a separate, tight-lidded, plastic culture dish containing an absorbent pad saturated with a holding medium and was held for 3 days at room temperature (approximately 22 C). Filters from the holding media were then transferred to m-fc agar medium. Bacterial colonies on the m-fc agar medium APPL. ENVIRON. MICROBIOL. were enumerated after incubation at 44.5 ± 0.2 C for 24 ± 2 h. Evaluation of holding media with pure fecal coliform cultures. Each holding medium was tested with the same 60 pure cultures. For m-vfc and m-st an additional 20 pure cultures were tested. Recovery for each holding medium was evaluated by first calculating the average of each quadruplicate set of results by DMF or IMF. The average DMF count was then compared with the average IMF count to provide a ratio (DMF/IMF). For each holding medium, the DMF/IMF ratio for each bacterial strain was used to calculate the arithmetic mean ratio, standard error of the mean, variance, and 95% confidence limits of the mean. Evaluation of holding media with natural water samples. Natural surface water and wastewater samples were collected from locations listed in Table 3 (see below). All were collected and transported to the laboratory in accordance with procedures described in Standard Methods (1). Each sample was tested in quadruplicate by IMF and by DMF with each of the three media, as described above, and evaluated by the same statistical procedures. Effect of length of delay on recovery of fecal coliforms from natural water samples held on m-st. Natural surface water and wastewater samples were collected from the locations listed in Table 5 (see below). All samples were collected and transported to the laboratory in accordance with procedures described in Standard Methods (1). Each sample was tested in quadruplicate by IMF on m-fc agar medium as described above and by DMF, with the sample held on m-st only for 1, 2, or 3 days before incubation. The results were evaluated statistically as described above. RESULTS Evaluation of holding media with pure cultures. The mean DMF/IMF ratio obtained with pure cultures showed that recovery of isolates held on m-st was significantly higher at the 95% confidence level than were recoveries obtained with the other two media (Table 2). Recovery with m-vfc was greater than that obtained with NSB. Evaluation of holding media with natural water samples. The mean DMF/IMF ratios obtained TABLE 2. Statistical comparison of recoveries from pure fecal coliform cultures held for 3 days before incubation No Holding DMF/IMF ratio Van- 95% confi medium meim cultures testeda (en (mean SM acedence limit ' SEM) ance of mean m-st ± m-vfc NSB a The same 60 pure cultures were used for all three media. Twenty additional pure cultures were used for m-st and m-vfc.

3 VOL. 46, 1983 TABLE 3. DELAYED INCUBATION FOR COLIFORM DETECTION 891 DMF/IMF ratios for 19 natural water samples held on m-st, m-vfc, or NSB for 3 days before incubation Date IMF/100 ml DMF/IMF Sample site (91 ma D (1981) (mean + SD) nm-st m-vfc NBS Albany County wastewater treatment plant Primary effluent 8/26 1,800,000 ± 300, Secondary effluent 8/26 6, Cayadutta Creek at Fonda 9/25 1,600 ± Delhi wastewater treatment plant secondary effluent 10/16 44,000 ± 3, Fish Creek at Schuylerville 9/ ± Hoosic River Petersburg 10/ ± Schaghticoke 10/9 35 ± State Line 10/2 27 ± Hudson River Bethlehem 10/21 1,900 ± Easton 10/9 390 ± Glenmont 9/23 3,700 ± Glens Falls 10/9 2,400 ± Route 4 (Thompson) 10/9 1, Route 197 (Fort Edward) 10/12 1,500 ± Stillwater 10/9 540 ± Waterford 9/23 3,800 ± Kayaderosseras Creek 10/ ± Mohawk River Fort Hunter 9/18 28 ± Cayadutta Creek 10/21 50 ± with natural water samples are shown in Table 3. A statistical evaluation of the mean DMF/IMF ratios (Table 4) showed that fecal coliform recovery with m-st was significantly higher at the 95% confidence level than were recoveries obtained with m-vfc or NSB. The recovery by DMF with m-st was also greater than the recovery obtained by IMF. Recoveries with m- VFC and NSB were not significantly different at the 95% confidence level. Effects of length of delay on recovery from natural water samples held on m-st. DMF/IMF ratios obtained with natural water samples and holding times of 1, 2, and 3 days on m-st are shown in Table 5. A statistical evaluation (Table 6) showed that the ratio increased daily over the 3-day period. Counts after 2 or 3 days of holding were significantly higher at the 95% confidence level than were IMF counts. Confirmation of fecal coliform colonies. A total of 641 fecal coliform colonies from m-fc test cultures from natural water samples were transferred to lauryl tryptose broth at 35 C and to EC medium at 44.5 C for verification (Table 7). Gas was produced in -94% of all EC tubes for each time interval tested. DISCUSSION An improved DMF will enable health authorities to make more reliable estimates of bacterial contamination in the natural aquatic environment and will aid in the identification of sources of contamination. Such a test may also permit bacteriological testing of recreational and other natural waters in areas where testing was previously not possible. Most coliform bacteria may become injured when exposed to natural waters for a few days (5, 11), and such injured bacteria may fail to grow on selective media unless resuscitation methods are used (7, 8). The results of our TABLE 4. Statistical comparison of recoveries of fecal coliforms from 19 natural water samples held for 3 days before incubation Holding DMF/IMF ratio Variance 95% confidence medium (mean ± SEM) limit of mean m-st m-vfc NSB

4 892 CHEN AND HICKEY APPL. ENVIRON. MICROBIOL. TABLE 5. DMF/IMF ratio for 15 natural water samples held on m-st for 1, 2, or 3 days before incubation Sample site Date IMF/100 ml DMF/IMF (1981) (mean + SD) 1 day 2 days 3 days East Greenbush wastewater treatment plant Primary effluent 10/28 2,700,000 ± 200, Primary effluent 12/7 790,000 ± 50, Secondary effluent 10/28 7,400 ± Belmont Lake, Long Island 10/ Cayadutta Creek, Fonda 10/ ± Hudson River Bethlehem 12/21 1, Brickyard, Albany 12/ Glenmont 12/18 1,400 ± Glens Falls 11/9 870 ± Lock 1, Troy 12/ ± Route 4 bridge (Waterford) 10/21 1,300 ± Route 197 bridge (east channel) 11/5 580 ± Route 197 bridge (west channel) 11/5 710 ± Thompson 11/6 680 ± Hoosic River, Route 346 bridge (Petersburg) 10/ ± TABLE 6. Statistical comparison of recoveries of fecal coliforms from 15 natural water samples held for 1, 2, or 3 days before incubation Delay DMF/IMF ratio Variance 95% confidence (days) (mean ± SEM) limit of mean ± ± TABLE 7. Percent confirmation of 641 typical blue fecal coliform colonies on m-fc medium held on m- ST % Confirmation with: Delay No. of colonies (days) Lauryl tryptose EC medium tested broth studies of pure cultures and natural waters demonstrate that fecal coliform counts obtained with m-st are significantly higher than those obtained with m-vfc or NSB. In addition, mean DMF recovery with m-st is higher than mean IMF recovery, and the difference increases daily with holding time up to 3 days. The increase may reflect a gradual resuscitation of sublethally injured organisms which could not be detected by the standard IMF. This hypothesis is supported by the observation that pure culture counts do not increase during holding. Moreover, in verification studies.94% of all presumed fecal coliform colonies obtained with the modified DMF were capable of producing typical fecal coliform reactions in lauryl tryptose broth and EC medium Ẇe therefore propose that m-st be substituted for m-vfc for use with the DMF fecal coliform test. ACKNOWLEDGMENT We thank D. Forrester for technical help. LITERATURE CITED 1. American Public Health Association Standard methods for the examination of water and wastewater, 15th ed. American Public Health Association, Inc., Washington, D.C. 2. Bordner, R. H., J. A. Winter, and P. V. Scarpino (ed.) Microbiological methods for monitoring the environment, water and wastes. EPA publication no. 600/ Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio. 3. Brodsky, M. H., B. W. Ciebin, and D. A. Schiemann Simple bacterial preservation medium and its application to proficiency testing in water bacteriology. AppI. Environ. Microbiol. 35: Geldreich, E. E., P. W. Kabler, H. L. Jeter, and H. F. Clark A delayed incubation membrane filter test for coliform bacteria in water. Am. J. Publ. Health 45: McFeters, G. A., S. C. Cameron, and M. W. LeChevalier Influence of diluents, media, and membrane filters on detection of injured waterborne coliform bacteria. Appl. Environ. Microbiol. 43: Panezai, A. K., T. J. Macklin, and H. G. Coles Coli-Aerogenes and Escheric hia coli counts on water samples by means of transported membranes. Proc. Soc. Water Treat. Exam. 14:

5 VOL. 46, 1983 DELAYED INCUBATION FOR COLIFORM DETECTION Speck, M. L., B. Ray, and R. B. Read, Jr Repair and enumeration of injured coliforms by a plating procedure. Appi. Microbiol. 29: Stuart, D. G., G. A. McFeters, and J. E. ScHUger Membrane filter technique for the quantification of stressed fecal coliforms in the aquatic environment. Appl. Environ. Microbiol. 34: Taylor, R. H., R. H. Bordner, and P. V. Scarplno Delayed-incubation membrane-filter test for fecal coliforms. Appl. Microbiol. 25: Toombs, R. W., and D. A. Connor Proficiency test sample media for single and mixed pure cultures of water pollution indicator bacteria. Appl. Environ. Microbiol. 40: Zaske, S. K., W. S. Dockins, and G. A. McFeters Cell envelope damage in Escherichia coli caused by shortterm stress in water. AppI. Environ. Microbiol. 40: