The Presence/Absence Coliform Test


 H2o Baja has a lab here in Cabo San Lucas.  After looking at several options for testing for total coliform and E. coli, we decided to use the Presence / Absense Coliform test.  Our supplier for the testing supplies is Hach Company, a world leader in testing equipment.  We have a very accurate German incubator and all the necessary apparatus to conduct many essential tests for drinking water quality as well as wastewater. At the moment, we are testing drinking water for total coliform and E. coli at no charge, so there is no reason not to stop in with your fresh water sample (under 2 hours is best).



The Presence-Absence Coliform Test for Monitoring Drinking Water Quality







Mr. Rice and Mr. Geldreich are with the Environmental Protection Agency (EPA). Mr. Rice is a Microbiologist in the Microbiological Treatment Branch, and Mr. Geldreich is EPA Senior Microbiologist. Ms. Read is a Senior Statistician with the Computer Sciences Corp., Cincinnati, OH.



The concern for improved monitoring of the sanitary quality of drinking water has prompted interest in alternative methods for the detection of total coliform bacteria. A simplified qualitative presence-absence test has been proposed as an alternate procedure for detecting coliform bacteria in potable water.


In this paper data from four comparative studies were analyzed to compare the recovery of total coliform bacteria from drinking water using the presence-absence test, the multiple fermentation tube procedure, and the membrane filter technique. The four studies were of water samples taken from four different geographic areas of the United States: Hawaii, New England (Vermont and New Hampshire), Oregon and Pennsylvania. The analysis of the results of these studies were compared, based upon the number of positive samples detected by each method. Combined recoveries showed the presence/absence test detected significantly higher numbers of samples with coliforms than either the fermentation tube or membrane filter methods, P< 0.01.


The fermentation tube procedure detected significantly more positive samples than the membrane filter technique, P<0.01. Based upon the analysis of the combined database, it is clear that the Presence-Absence test is as sensitive as the current coliform methods for the examination of potable water. The presence-absence test offers a viable alternative to water utility companies that elect to use the frequency-of-occurrence approach for compliance monitoring.


IN THE UNITED STATES, the total coliform group of bacteria serve as the principal microbiological parameter for determining the sanitary quality of drinking water. The National Interim Primary Drinking Water Regulations require that all public water supplies be monitored for coliform bacteria with the use of either the multiple fermentation tube (FT) procedure or the membrane filter (MF) technique (1). Proposed changes to existing regulations call for amending the recommended maximum contaminant level for coliform bacteria to 0 organisms per 100 ml and adopting a frequency-of-occurrence concept for compliance monitoring (2). This type of moni- 54 Public Health Report monitoring system would be used to establish a coliform compliance limit based on the fraction of samples containing coliform bacteria during a given period (3). This frequency-of-occurrence protocol, the presence-absence concept, would be used in place of the current system wherein compliance is based upon either the arithmetic average of coliform bacteria detected in water samples by the MF technique or the percentage of positive FTs found over a 30-day period. Such data can be obtained from conventional coliform tests (MF or FT) by translating any coliform count or positive tube results into a coliform occurrence. The concern for improved monitoring, especially for small water systems, has prompted interest in the use of apresence-absence (PA) procedure as an alternative method for detecting coliform bacteria in drinking water to determine compliance with a regulation based on the presence-absence concept.

One of the major concerns of monitoring drinking water for coliforms is to ensure that microbial flora or the different substances found in drinking water do not influence the results of detection methods. A search of the scientific literature indicates a paucity of information concerning the effect of geographic differences on coliform monitoring.

In this study, we explore the effect of geographic differences on the PA test in terms of coliform detection compared with MF and FT methods now

in use. The use of a simplified presence-absence approach for examining potable water was first proposed by Weiss and Hunter (4). A presence-absence procedure has been used extensively in the Province of Ontario, Canada (5), and has been compared with the MF technique (6). A PA procedure is currently listed as a tentative method for analyzing drinking water samples in “Standard Methods for the Examination of Water and Wastewater” (7). The PA test, unlike conventional methods, is a qualitative rather than a quantitative procedure. The procedure is a basic modification of the FT method and uses the same verification procedure. Unlike the FT procedure, 100 ml of sample are routinely analyzed. The procedure consists of inoculating the water sample into a bottle containing the appropriate concentration of PA medium (7) and a fermentation tube for gas entrapment. The bottle is incubated for 24 to 48 hours at 35 Celsius (C) and inspected for acid and gas production. If gas is noted in the fermentation tube or an acid reaction (as indicated by a color change of the indicator dye) is observed, a small inoculum of the culture is transferred to a tube of brilliant green lactose broth for confirmation. The production of gas in the confirmatory medium within 24 to 48 hours at 35C is related to coliform occurrence. The test is completed within a maximum time of 96 hours, and the results are reported as coliform present or absent.

The implementation of any new procedure for compliance monitoring requires the accumulation of a sufficient database to ensure that the procedure is comparable to the currently accepted methods. These data are especially important for the monitoring of potable water, where the vast majority of samples contain few or no coliforms (8). It is imperative, therefore, that any new procedure be able to detect very low numbers of coliform bacteria at a level of sensitivity equal to that of the procedures currently being employed. Recent research findings have indicated that the PA test is more sensitive than either the FT or MF procedures (9,10), while others have found the PA test to be at least as sensitive as the FT and MF procedures (8,11). The objective of our investigation was to evaluate the coliform data from four studies conducted in different geographic areas to determine the equivalency of the standard PA test with the conventional MF and FT procedures. Only those studies using currently acceptable protocols for coliform detection (7) are included in this analysis.



The analysis of the combined database shows the PA test to be very efficient for coliform detection in drinking water, with a level of recovery significantly higher than the other methods. Combined data show the PA test detected significantly more positive samples than either of the two standard coliform methods (FT and MF).


We conclude that the PA test possesses the sensitivity required for the detection of total coliform bacteria in potable water samples. This study indicates that the PA test is at least as sensitive as the current standard methods for detecting coliforms and that this evaluation holds true in various geographic regions in the United States. This test offers a viable alternative to water utility companies that elect to use the frequency-of-occurrence approach for compliance monitoring.

References …….

1. Environmental Protection Agency: National primary drinking water regulations. Federal Register 40: 59566, No. 248, pt. 141, Dec. 24, 1975. January-February 1989, Vol. 104, No. 1 57

2. Environmental Protection Agency: Drinking water; national primary drinking water regulation; total coliforms; proposed rule. Federal Register 52: 42224, No.212, pt. 141 and 142, Nov. 3, 1987.

3. Pipes, W. O., and Christian, R. R.: Estimation of mean coliform densities of water distribution system. J Am Water Works Assoc 76:60-64, November 1984.

4. Weiss, J. E., and Hunter, C. A.: Simplified bacteriological examination of water. J Am Water Works Assoc 31:707-713, April 1939.

5. Clark, J. A.: A presence-absence (P-A) test providing sensitive and inexpensive detection of coliforms, fecal coliforms and fecal streptococci in municipal drinking water supplies. Can J Microbiol 14:13-18, January 1968.

6. Clark, J. A.: The influence of increasing numbers of non-indicator organisms upon the detection of indicator organisms by the membrane filter and presence-absence tests. Can J Microbiol 26:827-832, July 1980.

7. Greenberg, A. E., Trussell, R. R., and Clesceri, L. S., editors: Standard methods for the examination of water and wastewater, Ed. 16. American Public Health Association, 1985.

8. Pipes, W. O., Minnigh, H. A., Moyer, B., and Trog, M. A.: Comparison of Clark’s presence-absence test and the membrane filter method for coliform detection in potable water samples. Appl Environ Microbiol 52:439-443, September 1986.

9. Jacobs, N. J., et al.: Comparison of membrane filter, multiple fermentation tube, and presence-absence techniques for detecting total coliforms in small community water systems. Appl Environ Microbiol 51:1007-1012, May 1986.

10. Caldwell, B. A., and Morita, R. Y.: Sampling regimes and bacteriological tests for coliform detection in groundwater. Project Report EPA/600/287/083. Environmental Protection Agency, Cincinnati, OH, 1987.

11. Fujioka, R., Kungskulniti, N., and Nakasone, S.: Evaluation of the presence-absence test for coliforms and the membrane filtration method for heterotrophic bacteria. In Proceedings of the Water Quality Technology Conference, American Water Works Association, Portland, OR, Nov. 16-20, 1986, pp. 271-279.

12. Fleiss, J. L.: Statistical methods for rates and proportions, Ed. 2. John Wiley and Sons, New York, 1981.

13. Rice, E. W., et al.: Comparison of media for recovery of total coliform bacteria from chemically treated water. Environ Microbiol 53:1571-1573, July 1987.