A Comparison of the Multiple-Tube Fermentation Method and the Colitag Method for the Detection of Waterborne Coliform Bacteria

Recreational water quality is an important issue in public health because of problems, including diarrhea, that are caused by fecal contamination. Constant monitoring for Escherichia coli (E. coli), an indicator organism for such contamination, is a way to reduce human exposure. This research project was designed to determine if the Colitag method for detecting E. coli is as reliable as the Multiple-Tube Fermentation (MTF) method, which is currently the technique approved by the U.S. Environmental Protection Agency. From September 2000 to March 2001, identical water samples were taken, approximately once a week, from Strawberry and Codornices Creeks, in Berkeley, California, to perform both tests simultaneously. The Colitag method was found to be as sensitive as the MTF method in detecting fecal coliforms in recreational water. Statistical analysis of parallel test results showed a strong linear correlation of 0.87 between the two methods, which held up well at both high and low fecal coliform counts. Because only 24 hours are required to obtain results from the Colitag method, compared to 96 hours for the MTF method, the former is preferable. Saving three days means that monitoring agencies could respond faster to sudden increases in E. coli and could therefore take immediate corrective action to ensure the public safety. Introduction Strawberry Creek, which runs through the campus of the university of California at Berkeley, represents an irreplaceable natural resource that is highly valued by both the university and the community at large. The riparian corridors provide essential places for educational, recreational, social, and individual activities (Charbonneau 1987). Therefore, the water quality of the creek is extremely important for human health, especially since the creek is used by many students for educational and recreational purposes, ranging from college students, who study it in Biology 1B at UC Berkeley (Moser 1999), to elementary school students, who come to play and picnic there (Charbonneau 1987). In past years, the water quality of the creek has caused great concern among members of the university community and their neighbors. An article in the student newspaper, the Daily Californian (July 2, 1973, p. 6), reported that fecal bacteria were contaminating the north fork of the creek from sources in the north Berkeley hills. This caused concern in the community because fecal bacteria can cause human disease and illness (Baker and Herson 1999). Another article in the paper, eight years later (November 2, 1981, p. 1), reported that the creek was being used as a sewer, contaminated by urban runoff, chemicals, and sewage. With recent efforts to improve the water quality of the creek, the levels of fecal coliform and Escherichia coli (E. coli) detected in the creek have decreased dramatically. Over the last century, scientists around the world have tried to come up with an easy, affordable, and accurate way of monitoring the quality of drinking and recreational water (Chang 2000, pers. comm.). One way to determine water quality is to measure the presence of E. coli, which is the main member of a large bacterial family Enterobacteriaceæ, which is composed of faculatively anaerobic Gram-negative rods that live in the gastrointestinal tract of most warmblooded animals (Edberg et al. 1997). The presence of E. coli in normal human intestines and feces has led to using the bacteria in nature as an indicator of fecal pollution in water (Edberg et al. 1997). Most E. coli strains are harmless, but several cause diarrhea. In severe cases, people, especially children and the elderly, have died from ingesting water or food contaminated with E. coli (AWWA Research Committee 1999). The current standard method for monitoring members of the coliform group in water is the Multiple-Tube Fermentation (MTF) technique, which is certified by the U.S. Environmental Protection Agency, USEPA (Clesceri et al. 1998). However, this method has the disadvantage of requiring up to 96 hours to obtain results. When a large quantity of pathogens is present in recreational water, it would be ideal to inform the public of this as soon as possible. But since the MTF technique is so slow, people may become sick from contact with recreational water before test results are released to them. Another disadvantage of this technique is that it is not E. coli specific, but instead measures all fecal coliforms. In 1986, USEPA recommended using E. coli as the indicator organisms instead of fecal coliforms for monitoring recreational water because the former give a more accurate measurement. However, this recommendation has not been widely accepted, since the MTF method has been around for almost 100 years as a way to measure fecal coliforms. When agencies are used to monitoring water with the same method for a long time, it is hard to get them to try new methods. The Colitag technique (Chang 2000, pers. comm.), on the other hand, has neither of these disadvantages. It detects only E. coli, and it yields results in only 24 hours. The purpose of the present research project, therefore, is to compare the results obtained from these two different methods to see if the Colitag method works as well in detecting fecal coliforms in recreational water as the MTF technique. Samples were taken not only from Strawberry Creek but also from Codornices Creek, which flows through UC Berkeleys Married Students Housing Village, in Albany. The second creek was added to the study because many children of all ages play nearby and may be at risk from contacting the water (Lee and Lee 2000). Another reason for testing this creek was to increase the range of fecal coliform levels tested by both techniques, because Codornices Creek has historically had higher counts of fecal coliform than Strawberry Creek (Maranzana 2000, pers. comm.). Methods Strawberry Creek is divided into two main branches, the north and south forks. The confluence of the two forks is located in Eucalyptus Grove at the western edge of the central campus, about 400 feet east of Oxford Street (Figure 1). Codornices Creek runs nearby UC Berkeleys Married Students Housing Village in Albany between Ninth Street and Gilman Street (Figure 1). Exactly the same sampling methods were used at Codornices Creek as at Strawberry Creek. The two sites were chosen because UC Berkeleys office of environment, health, and safety already uses them for weekly monitoring. Figure 1 Sampling Sites of Strawberry Creek at the confluence of North and South Forks and Codornices Creek at the intersection of Eighth and Gilman Streets Approximately every week between September 11, 2000 and March 5, 2001, two 100 ml samples of water were collected simultaneously in sterilized containers from each fork of Strawberry Creek (Figure 1). The samples were collected with this frequency in order to get a good variety of coliform counts from dry and wet seasons. In the past, high rainfall had usually been associated with high coliform counts in the creek water. The four samples were taken back to the Office of Environmental Health and Safety at UC Berkeley, which forwarded one north fork and one south fork sample to a commercial lab, Cerco Analytical Inc., in Pleasanton, California, to be analyzed by the MTF method (Clesceri et al. 1998, numbers 9221B [total coliforms] and 9221E [fecal coliforms]). The other two samples were analyzed by the Colitag method (fecal coliforms and E. coli). The controls used for the Colitag method were SaLT2, ECATCC 25922, ECTC 219, KpTC 249 and a blank, which consisted only of Colitag medium at 6.20 pH (Sloat & Ziel 1991). Colitag, which is an enzymatic indicator-based medium, contains o-nitrophenylß-dgalactopyranoside (ONPG), 4-methylumbelliferyl-ß-D-glucuronide (MUG), and other selective ingredients that are specific to coliforms with little interference from high heterotrophic bacteria counts. The fecal coliforms produced an enzyme to cleave the ONPG and release the indicator. A positive test for fecal coliforms was indicated by a yellow color. Failure to cleave with ONPG with little or no growth constituted a negative reaction and thus had a color similar to that of the blank. E. coli cleaves MUG and produces a blue fluorescence under long-wave ultraviolet (UV) light. Therefore, a UV lamp was used to read the results. An Indole test for E. coli was also conducted with the use of Kovacs reagent. A positive test was produced when a reddish surface layer appeared with a few drops of the Kovacs reagent due to E. coli cleaving with the