A Rapid, Specific Membrane Filtration Procedure for Enumeration of Enterococci in Recreational Water

Abstract

A two-step membrane filter (MF) method with mE medium, upon which the membrane must be incubated for 48 h and then transferred to a substrate medium to differentiate enterococci, is recommended by the U.S. Environmental Protection Agency to measure enterococci in fresh and marine recreational waters. The original mE medium was modified by reducing the triphenyltetrazolium chloride from 0.15 to 0.02 g/liter and adding 0.75 g of indoxyl β-d-glucoside per liter. The new MF medium, mEI medium, detected levels of enterococci in 24 h comparable to those detected by the original mE medium in 48 h, with the same level of statistical confidence. In addition, the use of mEI medium eliminated the need to transfer the membrane to a substrate medium to differentiate enterococci from other genera of the fecal streptococcal group. Colonies from mEI medium were examined to determine the rates of false-positive and false-negative occurrences. mEI medium had a false-positive rate of 6.0% and a false-negative rate of 6.5%. Interlaboratory testing of the MF method with mEI medium demonstrated that the relative reproducibility standard deviations among laboratories ranged from 2.2% for marine water to 18.9% for freshwater. The comparative recovery studies, specificity determinations, and multilaboratory evaluation indicated that mEI medium has analytical performance characteristics equivalent to those of mE medium. The simplicity of use and decreased incubation time with mEI medium will facilitate the detection and quantification of enterococci in fresh and marine recreational waters.

The use of enterococci as an indicator of fecal contamination of recreational water was recommended by the U.S. Environmental Protection Agency (13) in 1986. The recommendation was based on studies which demonstrated that enterococci had a strong direct relationship to swimming-associated illness in both marine water (3) and freshwater (7) environments. A two-step membrane filter (MF) procedure described by Levin et al. (11) was used to quantify enterococci in these studies and is the procedure recommended for measuring the quality of recreational water by the U.S. Environmental Protection Agency Ambient Water Quality Criteria for Bacteria—1986 (13).

The two-step MF procedure for enterococci requires 48 h of incubation of the MF at 41°C on a selective primary isolation medium (mE agar) followed by transfer of the MF to an in situ esculin-iron agar (EIA) substrate medium, which is incubated for 20 min at 41°C. Pink to red colonies on the MF that produce a brownish black precipitate on EIA are identified as enterococci. The brownish black precipitate formed on EIA is the result of the hydrolysis of esculin to glucose and coumarin by the enzyme β-glucosidase. Coumarin forms a black precipitate in the presence of ferric citrate. The selective characteristics of the primary isolation medium (mE agar) result from the addition of nalidixic acid, cycloheximide (Acti-Dione), and triphenyltetrazolium chloride (TTC) to the medium and the elevated incubation temperature of 41°C. Nalidixic acid inhibits gram-negative bacteria, cycloheximide inhibits fungi, and TTC (0.15 g/liter) differentiates enterococci from other gram-positive cocci and inhibits background organisms. The specificity of the medium was reported to be 10% false-positive and 11.7% false-negative (11).

In 1980, Dufour (6) described a medium, similar to that of Levin et al. (11), for use in a single-step, 24-h MF procedure to enumerate enterococci in marine water and freshwater. The medium contained nalidixic acid, cycloheximide, a reduced concentration of TTC, and indoxyl β-d-glucoside, a chromogenic cellobiose analog used in place of esculin in the primary medium of Levin et al. (11) to differentiate enterococci from fecal streptococci. The addition of indoxyl β-d-glucoside into microbiological media results in β-glucosidase-positive enterococci producing an insoluble indigo blue complex which diffuses into the surrounding media, forming a blue halo around the colony.

The present study was undertaken to (i) evaluate modifications to the commercially available base medium mE agar which would produce recovery of enterococci equivalent to that in the two-step, 48-h procedure in a single-step, 24-h procedure; (ii) determine the specificity of the modified medium (mEI medium) for enterococci; and (iii) determine, through collaborative study, the variability among laboratories using mEI medium for samples from various aquatic environments.

MATERIALS AND METHODS

Media.

The ingredients in and method of preparation of mE and mEI media are given in Table 1. The EIA substrate medium used in the two-step procedure was prepared according to the instructions of the manufacturer (Difco, Detroit, Mich.) and the 19th edition of Standard Methods for the Examination of Water and Wastewater (1). mE agar (Difco) served as the reference medium for the comparability segment of the study. mEI agar was evaluated in the specificity and reproducibility segments of the study.

TABLE 1.

Formulation and preparation of enterococcal media evaluated

Ingredient	Amt (g/liter) in:
	mE agara	mEI agar
Agar	15	15
Peptone	10	10
NaCl	15	15
Esculin	1	1
Yeast extract	30	30
Cycloheximide	0.05	0.05
Sodium azide	0.15	0.15
Indoxyl β-d-glucoside		0.75
Distilled water	1,000	1,000
Nalidixic acidb	0.24	0.24
TTCb	0.15	0.02

^aFormulation of Levin et al. (11). 

^bAdded aseptically after autoclaving at 121°C for 15 min. 

Samples.

To determine comparability and specificity, natural samples of river, lake, and stream waters, primary and secondary wastewater effluents, and marine waters were collected in sterile containers and kept at <10°C. All samples except for marine waters were analyzed within 4 h of collection. Marine water samples were analyzed within 2 h of arrival by express mail from the coastal collection points. To determine the variability among laboratories using mEI agar, split samples of three fecally contaminated surface waters, a nonchlorinated primary wastewater effluent, a chlorinated secondary wastewater effluent, and two marine waters contaminated with a nonchlorinated primary wastewater effluent were prepared by the National Exposure Research Laboratory, Cincinnati, Ohio, and transported at <10°C to 12 independent collaborative test laboratories. Each laboratory was instructed by the referee laboratory when to test the samples to ensure that all had the same holding time.

MF procedure.

Selection of sample volumes tested varied with the sample source and prior history of the source when known. Marine waters found to be free of enterococci on initial analysis were seeded with different primary or secondary effluents to provide various levels and types of enterococci. Each sample was filtered through a 47-mm-diameter, 0.45-μm-pore-size cellulose acetate MF grid (Millipore Corp., Bedford, Mass.) held in a stainless steel filtration unit. Each filtration series was begun with steam-sterilized filtration units. Triplicate volumes of each sample were membrane filtered, and the filters were transferred to the surface of each of the media and evaluated for recovery of enterococci. All plates were incubated at 41 ± 0.5°C for 24 or 48 h. When the portion of the water sample to be filtered was smaller than 10 ml, 20 to 30 ml of sterile dilution water was added to the funnel in the absence of a vacuum prior to the addition of the sample. The use of mE agar required transfer of the MF to EIA and further incubation at 41 ± 0.5°C for 20 min in order to differentiate and enumerate enterococci. Enterococci were enumerated directly on mEI agar plates. On mEI medium, colonies with a blue halo, regardless of other colony coloration, were identified as enterococcal colonies.

Comparison of methods.

Bland and Altman (2) discussed various approaches for comparing two methods, where one might replace the other if its measurements are sufficiently equivalent with respect to the intended use of the measurements. We used their suggested graphic approach, where the difference between the methods is plotted against the mean. The mean of the differences and the standard deviation of the differences were used to evaluate agreement of the two methods. The equivalence of the two methods was tested with a paired t statistic to examine the hypothesis of zero bias (12).

Specificity.

Specificity was evaluated by verifying a representative number of target and nontarget colonies. Verification of presumptive target and nontarget colonies was based on the API 20 Strep analytical profile system (Biomerieux, Vitek, Inc., Hazelwood, Mo.) and, when necessary, pigment production (4) plus growth at 10 and 45°C in brain heart infusion broth (Difco), growth in brain heart infusion broth with 6.5% NaCl, and growth on bile-esculin agar (Difco).

Interlaboratory variability.

Interlaboratory variability of the single-step, 24-h MF method with mEI agar as the assay medium was determined from data submitted by 14 collaborators at 12 laboratories. All reagents and materials except for rinse water were supplied by the referee laboratory. MF counts for the samples reported by the laboratories were converted to log (base 10) units and statistically analyzed by AOAC International-approved procedures for reproducibility standard deviation (S_R) and interlaboratory reproducibility standard deviation (RSD_R) (10).

RESULTS AND DISCUSSION

Method comparability.

Modification of the method currently used to monitor water quality for regulatory purposes could lead to significant bias or specificity differences, which might, in turn, appreciably affect the water quality limits set by statute. For instance, if a modification of a current method had a 20% positive bias, then the microbial limit would in effect be lowered by 16.7%. Similarly, if the specificity, i.e., the false-positive or false-negative rates, changed significantly, the microbial limit would be altered. Therefore, it is critical that differences between two methods to be used for the same purpose be minimized to the extent possible.

The results of the comparison of mE and mEI recoveries from 10 marine and 26 nonmarine water samples were evaluated by the approach recommended by Bland and Altman (2). Figure 1 shows the scatter of the differences between mE and mEI recoveries over a broad range of enterococcal densities. The mean difference between the 36 paired samples, where the mEI value was subtracted from the mE value, was −1. This slight bias favored the mEI procedure, but when the mean difference was evaluated to determine if it was significantly different from 0 in a paired t test (11), it was found that the −1 mean did not differ from 0 more than can reasonably be expected by chance (P > 0.05; df, 35). The 95% confidence limits (limits of agreement) of the mean difference were ±12.6 colonies. These confidence limits are well within the sampling error of MF data, which is beyond control of the analyst (9). The results of this method comparison indicated that the methods agreed sufficiently closely that mEI agar can be substituted for mE agar in the MF procedure for measuring enterococci in recreational waters.

FIG. 1.

Comparison of 36 paired enterococcal assays on mE agar and mEI agar. Symbols: ○, marine water; •, freshwater. CL, confidence limits.

Specificity.

The specificity of mEI medium in the single-step, 24-h MF procedure was examined by determining (i) the percentage of typical colonies which were not verified as members of the enterococcal group (false-positives) and (ii) the percentage of all verified colonies that did not react typically (false-negatives). The 361 target and nontarget colonies examined were isolates from seawater collected on the east and west coasts of the United States and freshwater samples from three states. A total of 94% (187 of 199) of the verified target colonies and 6.5% (13 of 200) of all verified target and nontarget colonies did not react typically (false-negatives) as enterococci. These rates are an improvement over the specificities of 90% confirmed positives and 11.7% false-negatives reported for mE agar (3).

Multilaboratory variability.

The focus of this part of the study was to examine the interlaboratory reproducibility of the 24-h MF method with mEI medium. Fourteen collaborators at 12 laboratories examined seven split water samples. Results that were found to be aberrant outliers by the single and double Grubbs tests (10) were not included in the statistical analysis. Table 2 shows the means and statistical summary of the results reported by the collaborators for the four types of water samples examined. The S_R was obtained by calculating the standard deviation of all the data because there were no replicate determinations.

TABLE 2.

Variability among laboratories using the mEI procedure for enumerating enterococci in various types of water

Water type	No. of labora- tories	Geometric mean (CFU/ 100 ml)	Log geometric mean	SR	RSDR (%)
Fresh	12	10.2	1.008	0.191	18.9
	12	173.8	2.240	0.135	6.0
Marine	10	4,487	3.652	0.080	2.2
Chlorinated secondary effluent	11	3,069	3.487	0.171	4.9
Nonchlorinated primary effluent	10	337.3	2.528	0.077	3.0

Reproducibility among laboratories (RSD_R) for freshwater, marine water, chlorinated secondary effluent, and nonchlorinated primary effluent ranged from 2.2% for marine water to 18.9% for freshwater with a low enterococcal density. The greater variation (18.9%) for freshwater with low enterococcal counts (10 or less per 100 ml) is attributable to the normal increased sampling variability found with low-count samples. The reported among-laboratory reproducibility (precision) for the enumeration of enterococci in water with mEI agar closely matches the precision (RSD_R) for the enumeration of coliforms on dry rehydratable films of 6.9 to 22.4% for dairy products (5) and greatly exceeds the precision (RSD_R) for the enumeration of Clostridium perfringens in nonchlorinated wastewater, sediment, and marine, surface, and drinking waters of 24 to 41% (8).

mEI medium preparation.

To provide information on the heat stability of indoxyl β-d-glucoside in medium preparation, 16 comparative assays of surface and marine waters were performed with two sets of mEI medium. One set was prepared with indoxyl β-d-glucoside added before sterilization, and the other set was prepared with indoxyl β-d-glucoside added after sterilization. Nalidixic acid and TTC were added to both sets of medium after sterilization. Cycloheximide is an ingredient of mE agar base. Results revealed that indoxyl β-d-glucoside is resistant to the heat of the sterilization process (121°C for 15 min) and thus can be added prior to sterilization, making the preparation of mEI medium easier and quicker.