SANTACRUZ...SANTACRUZ CITY OF OFFICE OF THE CITY MANAGE R 809 Center Street, Room I 0, Santa Cruz, 95060 (831) 420-5010 Fax: (831) 420-501•

C I T Y O F

SANTACRUZ �

OFFICE OF T H E CITY M A N AGE R

809 Center Street, Room I 0, Santa Cruz, CA 95060 • (831) 420-5010 • Fax: (831) 420-5011 • www.cityofsanlacruz.com

Dr. Jean-Pierre Wolff California Regional Water Quality Control Board Central Coast Region Pretreatment Programs Section 895 Aerovista Place, Suite 101 San Luis Obispo, CA 93401-7906 [email protected]

December 1, 2016

RE: DECEMBER 9, 2016 WATER BOARD AGENDA ITEM 16 - ATTACHMENT 2 -303(d)

Dear Dr. Wolff

The City of Santa Cruz wishes to address the Board meeting of December 9, 2016 in respect to the proposal to list several local beaches for impairment on the basis of Total Colifonn and Fecal Coliform data values derived from data compiled from the Beach Watch program. The City's presentation will be based upon the provisions of Chapter 4 of the Water Quality Control Policy For Developing California's Clean Water Act Section 303(d) List as accessed at: http://www.waterboards.ca.gov/water _ issues/programs/tmdl/3 03d _listing.shtml .

We request up to 10 minutes to apprise the Board of the available data that more effectively characterizes the water quality at these beaches, as well as recent infrastructural investments that are believed to have contributed to the better water quality measurements at these beaches. The local beaches adversely affected by the proposal, and as listed in the attachment are:

1. Pacific Ocean at Cowell Beach for Fecal and Total Coliform 2. Pacific Ocean at Main Beach for Total Coliform 3. Pacific Ocean at Main Beach at San Lorenzo River Mouth for Total Coliform 4. Pacific Ocean at Mitchells Cove Beach for Total Coliform 5. Pacific Ocean at Natural Bridges Beach for Total Coliform 6. Pacific Ocean at Seabright (Castle) Beach for Total Coliform

The presentation of data on Total Coliforms for these beaches is based upon analyses performed with microbiological methods more suitable to assaying total coliform counts in marine

Dr. Jean-Pierre Wolff December I, 2016 Page 2

environments. The results will show that where these have been applied the Total Coliform numbers are in compliance with the Shellfish Harvesting Limits.

The presentation will highlight data indicating the compliance with the Fecal Coliform limits at Cowell Beach.

The presentation will offer data trends that more accurately reflect the Fecal and Total Coliform status at Cowell Beach and adjacent waters since infrastructural improvements were made at and near the Wharf.

Finally the City will be recommending that the Board direct that these beaches be reclassified to either of the alternative statuses, as appropriate, including "Do Not List" pending the thorough review of better quality information.

If you have any questions, please contact the City's Laboratory and Environmental Compliance Manager, Akin Babatola, at 831-420-6045 or [email protected] .

Sincerely,

Maitin Bernal City Manager

cc: Mary Hamilton, [email protected]

Mark Dettle, Director Public Works Mauro Garcia, Director Parks and Recreation

P:\CMAD\ Word(Wpfiles)\S UZANNEU\CM Bernal\Leuers\ Wolff.docx

∗ Purpose: Offer a data-driven alternative to the proposed listing of City beaches for impairments based upon chapter 4 of the WQC Policy developing California’s CWEA 303(d) listing. (http://www.waterboards.ca.gov/board_decisions/adopted_orders/resolutions/2015/020315_8_amendment_clean_version.pdf)

∗ Identify implicit error in processing Beach Watch data to this process on such a scale;

∗ Highlight avoidable economic and social damage from the avoidable error; ∗ Identify appropriate methodologies for supporting the appropriately

stringent purpose of shellfish harvesting coliform limits ∗ Identify why defined substrate method allowed under Beach Watch is

inappropriate in this environment ∗ Present long term data on a select beach where the methods were applied

CWA 303(d) listing: City of Santa Cruz Beaches- alternative data-driven

proposal

1 / 21 Item No. 16 Presentation December 8-9, 2016

City of Santa Cruz Presentation Powerpoint

http://www.waterboards.ca.gov/board_decisions/adopted_orders/resolutions/2015/020315_8_amendment_clean_version.pdf








∗ Current requirement for Shellfish harvesting Limit: ∗ median < 70 MPN/ 100 ml ∗ (and 10% > 230)



This image cannot currently be displayed.



∗ All of the following tables are compiled from analyses in Membrane Filtration using Standard Method SM9222B at the City of Santa Cruz Environmental Laboratory ELAP CA 1176

∗ All analyses were performed either in support of: ∗ routine sanitary surveys; ∗ investigations by the Environmental Compliance program ∗ other City mandated programs. Therefore the limited range of beaches sampled, and the number of data points. However they provide truly accurate and diagnostic information as to the water quality at these beaches.



∗ All of the tables present the median values of the Total Coliform counts to be assessed against the NSSP (National Shellfish Sanitation Program) Total Coliforms standards: ∗ median < 70 MPN/ 100 ml ∗ (and 10% > 230)



Natural Bridges Total Coliform Counts (CFU/100mL) Sample Date Counts Running Medians 8/26/2013 <5 9/3/2013 5 9/9/2013 10

9/16/2013 <5 11/18/2013 12 10 11/25/2013 24 11 12/2/2013 4 11 12/9/2013 8 10

12/16/2013 8 8 2/24/2014 8 8 3/10/2014 12 8 3/17/2014 28 8 3/24/2014 8 8 3/31/2014 4 8 4/7/2014 4 8

Median 8



Mitchell's Cove Total Coliform Counts (CFU/100mLs) Sample Date Counts Running Median

2-Aug-10 4 9-Aug-10 10

16-Aug-10 19 23-Aug-10 2 30-Aug-10 1 10-Jan-11 12 24-Jan-11 5 5 31-Jan-11 5 5 7-Feb-11 2 5

14-Feb-11 280 5 22-Feb-11 10 5 28-Feb-11 6 6 7-Mar-11 4 6

16-May-11 2 6 23-May-11 2 4

6-Jun-11 21 4 7-Jun-11 33 4

13-Jun-11 31 21 21-Jun-11 5 21 27-Jun-11 8 21 1-Aug-11 2 8




8-Aug-11 6 6 15-Aug-11 8 6 22-Aug-11 6 6 30-Aug-11 10 6 6-Sep-11 2 6

12-Sep-11 50 8 27-Sep-11 2 6 30-Sep-11 27 10 3-Oct-11 10 10

10-Oct-11 18 18 17-Oct-11 14 14 24-Oct-11 94 18 31-Oct-11 4 14 7-Nov-11 17 17 5-Mar-12 122 17

13-Mar-12 12 17 19-Mar-12 10 12 26-Mar-12 32 17 2-Apr-12 32 32 9-Apr-12 20 20

16-Apr-12 4 20




23-Apr-12 180 32 30-Apr-12 16 20 7-May-12 16 16 27-Aug-12 2 16 4-Sep-12 12 16

10-Sep-12 12 12 1-Oct-12 120 12 8-Oct-12 30 12

25-Feb-13 8 12 4-Mar-13 8 12

11-Mar-13 24 24 18-Mar-13 12 12 25-Mar-13 14 12 1-Apr-13 168 14 8-Apr-13 4 14

13-May-13 42 14 20-May-13 18 18 28-May-13 3,950 42

3-Jun-13 68 42 10-Jun-13 84 68 17-Jun-13 8 68




24-Jun-13 4 68 5-Aug-13 68 68

12-Aug-13 4 8 19-Aug-13 8 8 26-Aug-13 5 5 3-Sep-13 15 8 9-Sep-13 5 5

16-Sep-13 5 5 18-Nov-13 4 5 25-Nov-13 32 5 2-Dec-13 32 5 9-Dec-13 8 8 24-Feb-14 36 32 3-Mar-14 33 32

10-Mar-14 104 33 17-Mar-14 32 33 24-Mar-14 8 33 31-Mar-14 32 32 7-Apr-14 8 32 Median 12



∗ Previous slides have shown data from: ∗ Pacific Ocean at Natural Bridges with MF Total Coliform counts

and medians well below the Shellfish Harvesting limits ∗ Pacific Ocean at Mitchell’s Cove with MF Total Coliform counts

and medians well below the Shellfish Harvesting limits



∗ The special case of Cowell Beach for Fecal and Total Coliform relies upon:

∗ Geographic precision ∗ Affected area of the Beach was/is limited and cannot be fairly used

to characterize Cowell Beach ∗ Affected area has been improved with significant infrastructural

investments

∗ Analytic precision ∗ Results are validated in profiles developed from measurements;

and are briefly presented here







Geometric Mean (5 sampling events) of Total Coliforms, Fecal Coliforms and Enterococcus for Cowell and Main Beach during August and September 2015

City of Santa Cruz Environmental Compliance and Environmental Laboratory

The geometric mean is a type of mean or average, which indicates the central tendency or typical value of a set of numbers by using the product of their values. The use of a geometric mean "normalizes" the ranges being averaged, so that no range dominates the weighting.

Samples for bacteria were collected weekly for 5 weeks along Cowell and Main Beach with roughly 75 feet between sampling locations.

The highest concentrations were measured at the Wharf with decreasing concentrations to the East and West.



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Average Indicator Bacteria values at Wharf-West - City of Santa Cruz Environmental Laboratory

Enterococci - Membrane Filtration

Fecal Coliforms - Membrane Filtration

Total Coliforms - Membrane Filtration





∗ Recommendations consistent with chapter 4 of the controlling document:

∗ Allow the review of data generated from matrix appropriate methodologies in marine environment, currently not required and NOT the source of the current data;

∗ Allow for more time to generate these appropriate data before reaching a finding;

∗ Current proposals are based upon Total Coliform data derive from Colilert analyses.

∗ Following slides summarize the difficulties of using those data for these purposes (Shellfish Harvesting Protection purposes).



∗ •https://www.cdph.ca.gov/HealthInfo/environhealth/water/Documents/Beaches/RecommendedMethodsforAB411.pdf

∗ Stipulates that: Colilert 18 is NOT recommended for the enumeration of total coliforms from marine water.

Published studies suggest there are substantial false positives, yielding higher total coliform counts from marine water. However, it is recognized that this method is easy to use, gives rapid and sensitive results, and has greater precision, when used for quantitative information, than the multiple tube fermentation test. • If Colilert 18 is to be used for AB 411 monitoring for total coliforms it must be acknowledged that this method may result in an overestimation of true total coliform numbers, which errors in favor of protecting public health. Total coliform results that repeatedly exceed the AB 411 standards should be verified with a more conservative method.



https://www.cdph.ca.gov/HealthInfo/environhealth/water/Documents/Beaches/RecommendedMethodsforAB411.pdf








∗ 1. Marine Bacteria Cause False-Positive Results in the Colilert-18 Rapid Identification Test for Escherichia coli in Florida Waters

∗ John M. Pisciotta,1 Damon F. Rath,2 Paul A. Stanek,2 D. Michael Flanery,2 and Valerie J. Harwood1* ∗ Department of Biology, University of South Florida, Tampa, Florida 33620-5150,1 and Pinellas County Health Department, ∗ Florida Department of Health, Clearwater, Florida 337642

∗ Received 9 August 2001/Accepted 5 November 2001APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2002, p. 539–544 Vol. 68, No. 2 0099-2240/02/Copyright ©

∗ 2. False-positive coliform reaction mediated by Aeromonas in the Colilert defined substrate technology system: Letters in Applied Microbiology (1998) JPB Landre, et. al.



∗ Thanks to the Board for the opportunity to present and ∗ To Water Board staff for assistance and cordiality through the

process.

∗ City of Santa Cruz, California ∗ Akin Babatola ∗ Laboratory & Environmental Compliance Manager



California Beaches—Regulations and Guidance Page 1 of 4

The following methods are recommended by the Environmental Laboratory Accreditation Program (ELAP) and the Microbiological Disease Laboratory (MDL) for the analysis of recreational marine water for compliance with Health and Safety Code §115880 [Assembly Bill 411 (AB 411), Statutes of 1997, Chapter 765]. Recommended Methods for the Analysis of Recreational Marine Water for AB 411 TOTAL COLIFORM BACTERIA

• Total Coliform by Multiple Tube Fermentation (MTF) — SM 9221 B (1,2) • Total Coliform by Membrane Filtration (MF) Using m-Endo — SM 9222 B (1,2)

See comments below on ColilertTM 18 Medium (IDEXX) (Quanti-TrayTM) for total coliforms FECAL COLIFORM BACTERIA

• Fecal Coliform* by Multiple Tube Fermentation (MTF) Using EC Medium — SM

9221 C, E (1,2) • Fecal Coliform* by Membrane Filtration Using m-FC (7,8) — SM 9222 D (1,2)

______ * With the written approval of the local health officer and with data showing comparative numbers for fecal coliforms and E. coli, a laboratory may instead test for E. coli, a subset of fecal coliforms (US EPA’s definition), using ColilertTM 18 Medium (IDEXX). Guidance on comparative testing is available in DHS Salt Water Beaches Guidance. Comparative testing must be performed only by laboratories certified by ELAP for the methods being compared. Laboratories must retain the results of the parallel testing in their files, consistent with their record retention procedures, and must make these data available for review upon request by the State. ENTEROCOCCUS BACTERIA

• Enterococci by Membrane Filtration (MF) Using mE (9) or mEI (11)— SM 9230 C (1,2); EPA Method 1600 (10)

• Enterococci by EnterolertTM (3,4,5) — IDEXX Co. JUSTIFICATION:

• The US EPA in its publication Ambient Water Quality Criteria for Bacteria –1986 (13) has recommended the use of E. coli or enterococci for testing fresh waters and enterococci only for marine waters.

• AB 411 and its implementing regulations require that marine water be tested for total coliforms, fecal coliforms and enterococci.

• Testing for three groups of indicator organisms with traditional methods for water testing requires much staff time, media and equipment space. Streamlined test methods with fast turnaround times and acceptable data are desired.

Item No. 16 Presentation Attachment 1 December 8-9, 2016

1. RecommendedMethodsforAB411.pdf

http://www.cdph.ca.gov/HealthInfo/environmentalhealth/water/Pages/Beaches.aspx


• Because of these concerns, ELAP and MDL reviewed the published literature to see if we could justify the use of two rapid methods using chromogenic/ fluorogenic substrates, one that identifies both total coliforms and E. coli (5,10) and the other, enterococci.(3,4,5)

• A number of laboratories have indicated a desire to test for E. coli in place of fecal coliforms. In US EPA’s Action Plan for Beaches and Recreational Waters

(14), E. coli is defined as "a subset of the fecal coliform group that is part of the normal intestinal flora in humans and animals and is, therefore, a direct indicator of fecal contamination of the water." E. coli is considered to be a more specific indicator of fecal contamination.(6,13)

• ColilertTM 18 Medium (IDEXX) (Quanti-TrayTM) is being used by these laboratories to report E. coli in place of fecal coliforms.

• Each method has its advantages and its shortcomings. Since California is examining marine water for three groups of indicator organisms, it is felt that, based on EPA’s definition of E. coli, public health will not be compromised if ELAP is flexible on the substitution of E.coli for fecal coliforms.

• Colilert 18 is not recommended for the enumeration of total coliforms from marine water. Published studies suggest there are substantial false positives, yielding higher total coliform counts from marine water. (5,12) However, it is recognized that this method is easy to use, gives rapid and sensitive results, and has greater precision, when used for quantitative information, than the multiple tube fermentation test.

• If Colilert 18 is to be used for AB 411 monitoring for total coliforms it must be acknowledged that this method may result in an overestimation of true total coliform numbers, which errors in favor of protecting public health. Total coliform results that repeatedly exceed the AB 411 standards should be verified with a more conservative method. Such tests must be performed only by laboratories certified by ELAP for the method.

• EnterolertTM medium in Quanti-TraysTM (IDEXX) is a 24-hour method for enterococci. Published literature supporting the use of this medium is available

(3,4,5). The medium is approved for use in some states for the testing of marine recreational water.

Review of Available Methods: Total Coliforms Multiple Tube Fermentation (MTF)

• Pros: Much historical data. Substantial scientific support. Allows testing of all kinds of waters, including colored and turbid.

• Cons: Requires up to 4 days for completion. Requires increase in tubes, media, incubator space, labor and time. Imprecision of MPN enumeration. 95% confidence limits are broad.

Membrane Filtration (MF)



ABabatola

Highlight


• Pros: Substantial scientific support. Much historical data. A direct count of organisms. Greater precision and accuracy.

• Cons: Can require up to 3 days for completion. Verification of colonies required. May not pick up viable but stressed organisms. Not all waters can be filtered. Technically more complex than other methods. Labor intensive.

ColilertTM 18 Quanti-Tray

• Pros: Easy to use. More sensitive. Results in 24 hrs. Greater precision in quantitation compared with MTF (5 tube mpn) Less staff time, media and incubator space required.

• Cons: Substantial false positives with marine water yielding higher counts than MTF method.(12)

Fecal Coliforms or E. coli Multiple Tube Fermentation (MTF) for fecal coliforms

• Pros: Much historical data. Substantial scientific support. Allows testing of all kinds of waters, including colored and turbid.

• Cons: Requires up to 4 days for completion. Requires increase in tubes, media, incubator space, labor and time. Not all thermal tolerant fecal coliforms are E. coli .(1,2) Imprecision of MPN enumeration. 95 % confidence limits are broad.

Membrane Filtration (MF) for fecal coliforms

• Pros: Much historical data. Substantial scientific support. Provides direct count of organisms.

• Cons: Can require up to 3 days for completion. Verification of colonies required. Not all waters can be filtered. Technically more complex than other methods. Not all thermal tolerant fecal coliforms are E.coli.

ColilertTM 18 Quanti-TrayTM for E. coli

• Pros: Easy to use. Results in 24 hrs. Greater precision in quantitation compared with MTF. Sensitive. E. coli is a (usually major) subset of fecal coliforms.

• Cons: Not listed as one of the organisms required by AB 411. Not all E. coli are mug positive – false negatives; not all fluorescent organisms are E. coli – false positives.(12)

June Kani, ELAP, and Dan Mills, Ph.D., MDL 3/24/00 References 1. American Public Health Association. 1992. Standard Methods for the Examination of Water and Wastewater, 18th ed. American Public Health Association, Washington DC.




2. American Public Health Association. 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed. American Public Health Association, Washington DC. 3. Abbott, S., B. Caughley, and G. Scott. 1998. Evaluation of Enterolert for the Enumeration of Enterococci in the Marine Environment. New Zealand Jour. of Marine & Freshwater Research. 32: 505-513. 4. Budnick, G.E., R.T. Howard and D.R. Mayo. 1996. Evaluation of Enterolert for Enumeration of Enterococci, In Recreational Waters. Appl. Environ. Microbiol. 62: 3881-3884. 5. Eckner, K.F. 1998. Comparison of Membrane Filtration and Multiple-Tube Fermentation by the Colilert and Enterolert Methods for Detection of Waterborne Coliform Bacteria, Escherichia coli and Enterococci Used in Drinking and Bathing Water Quality Monitoring in Southern Sweden. Appl. Environ. Microbiol. 64: 3079-3083. 6. Fujioka, R.S. Indicators of Marine Recreational Water Quality, Ch. 18 in: Manual of Environmental Microbiology. Hurst, C.J., G.R. Knudson, J.J. McInerney, L.D. Stetzenbach, and M.V. Water, Eds. ASM Press, Washington D.C. pp. 176-183. 7. Geldreich, E.E., H.F. Clark, C.B. Huff, and L.C. Best. 1965, Faecal-coliform Organism Medium for the Membrane Filter Technique. J. Am. Water Works Assoc. 57: 208-214. 8. Green, B.L., W. Litsky and K.J. Sladek. 1980. Evaluation of Membrane Filter Methods for Enumeration of Faecal Coliforms from Marine Water. Marine Environmental Research 3: 267-276. 9. Levin, M.A., J.R. Fischer and V.J. Cabelli. 1975. Membrane Filtration Technique for Enumeration of Enterococci in Marine Waters. Applied Microbiology 30: 66-71. 10. Messer, J.W. and A.P. Dufour. 1997. Method 1600: Membrane Filter Test Method for Enterococci in Water. US EPA Office of Water, Washington, DC. EPA-821-R-97_004. 11. Messer, J.W. and A.P. Dufour. 1998. A Rapid, Specific Membrane Filtration Procedure for Enumeration of Enterococci in Recreational Water. Appl. Environ. Microbiol. 64: 678-680. 12. Palmer, C.J., Yu-Li Tsai, A.L. Lang, and L.R. Sangermano. 1993. Evaluation of Colilert-Marine Water for Detection of Total Coliforms and Escherichia coli in the Marine Environment. Appl. Environ. Microbiol. 59: 786-790. 13. US Environmental Protection Agency, Office of Water, 1986. Ambient Water Quality Criteria for Bacteria – 1986. EPA 440/5-84-002, Washington DC. 22 pp. 14. US Environmental Protection Agency, Office of Water, 1999. Action Plan for Beaches and Recreational Waters, EPA/600/R-98/079, March 1999, 19 pp



APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2002, p. 539–544 Vol. 68, No. 20099-2240/02/$04.00�0 DOI: 10.1128/AEM.68.2.539–544.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Marine Bacteria Cause False-Positive Results in the Colilert-18 RapidIdentification Test for Escherichia coli in Florida Waters

John M. Pisciotta,1 Damon F. Rath,2 Paul A. Stanek,2 D. Michael Flanery,2 and Valerie J. Harwood1*Department of Biology, University of South Florida, Tampa, Florida 33620-5150,1 and Pinellas County Health Department,

Florida Department of Health, Clearwater, Florida 337642

Received 9 August 2001/Accepted 5 November 2001

The Colilert-18 system for enumeration of total coliforms and Escherichia coli is approved by the U.S.Environmental Protection Agency for use in drinking water analysis and is also used by various agencies andresearch studies for enumeration of indicator organisms in fresh and saline waters. During monitoring ofPinellas County, Fla., marine waters, estimates of E. coli numbers (by Colilert-18) frequently exceeded fecalcoliform counts (by membrane filtration) by 1 to 3 orders of magnitude. Samples from freshwater sites did notdisplay similar discrepancies. Fecal coliforms, including E. coli, could be cultured from 100% of yellowfluorescent wells (denoting E. coli-positive results) inoculated with freshwater samples but could be culturedfrom only 17.1% of the “positive” wells inoculated with marine samples. Ortho-nitrophenyl-�-D-galactopy-ranoside (ONPG)-positive or 4-methylumbelliferyl-�-D-glucuronide (MUG)-positive noncoliform bacteria werereadily cultured from Colilert-18 test wells inoculated with marine samples. Filtered cell-free seawater did notcause false positives. Coculture preparations of as few as 5 CFU of Vibrio cholerae (ONPG positive) andProvidencia sp. (MUG positive) ml�1 inoculated into Colilert-18 caused false-positive E. coli results. Salinityconditions influenced coculture results, as the concentration of coculture inoculum required to cause falsepositives in most wells increased from about 5 CFU ml�1 in seawater diluted 1:10 with freshwater to �5,000CFU ml�1 in seawater diluted 1:20 with freshwater. Estimated E. coli numbers in various marine water samplesprocessed at the 1:10 dilution ranged from 10 to 7,270 CFU·100 ml�1, while E. coli numbers in the samesamples processed at the 1:20 dilution did not exceed 40 CFU·100 ml�1. The lower estimates of E. coli numberscorresponded well with fecal coliform counts by membrane filtration. This study indicates that assessment ofE. coli in subtropical marine waters by Colilert-18 is not accurate when the recommended 1:10 sample dilutionis used. The results suggest that greater dilution may diminish the false-positive problem, but further study ofthis possibility is recommended.

Coliform bacteria are widely used as indicators of fecal con-tamination of both fresh and marine waters. Certain membersof the coliform group live outside of the gastrointestinal tractin the environment and may create a false indication of fecalcontamination. A more specific indicator of fecal contamina-tion is Escherichia coli, a thermotolerant fecal coliform bacte-rium distinguished by its ability to grow at 44.5°C and by itsexpression of the enzyme �-D-glucuronidase. This enzyme hy-drolyzes 4-methylumbelliferyl-�-D-glucuronide (MUG) toform a fluorescent (F) product that is visible with UV light (11,23) and that is used to differentiate E. coli from other fecalcoliforms.

The Colilert-18 defined-substrate technology system (IdexxLaboratories, Inc., Westbrook, Maine) is intended to providerapid (18-h), standardized quantitation of total coliforms andE. coli. Various versions of the Colilert system were shown toyield results that are statistically consistent with the standardmethods of membrane filtration and multiple-tube fermenta-tion testing used for the detection of coliforms and E. coli infreshwater (2, 3, 6, 7). The U.S. Environmental ProtectionAgency has approved Colilert for use in drinking water mon-

itoring (9, 10), and analyses of E. coli in fresh (6, 12, 21) andsaline (18, 22) natural waters have been published.

The Colilert-18 assay system is based on a defined substratemedium containing MUG and ortho-nitrophenyl-�-D-galacto-pyranoside (ONPG) and is used for the one-step detection ofboth total coliforms and E. coli in water samples. ONPG is acolorless lactose analog that is hydrolyzed by �-D-galactosi-dase, an enzyme common to all coliforms, to form a yellow (Y)product. �-D-Glucuronidase is present in relatively few bacte-rial species but is found in most E. coli strains. In the Colilertsystem, a Y color change following incubation is indicative oftotal coliforms, while a Y reaction in conjunction with fluores-cence under UV light is considered indicative of E. coli. A mostprobable number (MPN) estimate of total coliform and E. colinumbers is achieved though use of a plastic Quantitray parti-tioned into 48 small (120-�l) and 49 large (1.6-ml) wells.

Discrepancies, including E. coli false positives and false neg-atives, have been published (6, 8, 16, 17) for various Colilertformulations. It has been reported that up to 34% of E. colistrains in human feces (1) and 10 to 20% of E. coli strainsisolated from environmental sources (21) are not MUG posi-tive. These isolates would be false negatives in the Colilertsystem. Organisms, including Aeromonas spp. (16), pseudo-monads (14), some Salmonella and Shigella spp. (15, 20), andFlavobacterium spp. (19), can produce MUG-positive reac-tions, which may lead to false-positive results if the organismsare also lactose positive or are growing in a mixed culture with

* Corresponding author. Mailing address: Department of Biology,SCA 110, University of South Florida, 4202 E. Fowler Ave., Tampa,FL 33620. Phone: (813) 974–1524. Fax: (813) 974–3263. E-mail:[email protected].

5391 / 6 Item No. 16 Presentation Attachment 2

December 8-9, 2016 2. Colilert overestimates marine coliform bacteria

lactose-positive bacteria. It has been noted that the efficacy ofthe test should first be evaluated for source waters, such asstorm runoff, wastewater effluent, or marine waters (7), yet themajor work on the quantitative accuracy of Colilert assay sys-tems for E. coli in U.S. marine waters was carried out exclu-sively in California waters (18). Marine waters in California aremuch colder than subtropical waters, and the dominant au-tochthonous bacteria would therefore be expected to differfrom those of warmer waters. No studies, to our knowledge,have compared the accuracy of Colilert to that of standardenumeration techniques for indicator bacteria in tropical orsubtropical waters. In spite of this paucity of information, theColilert-18 system is frequently used by local and state agenciesand by other entities for water quality monitoring in fresh andsaline waters.

This study was prompted by the observation of high E. colicounts (103 to 104 CFU·100 ml�1) by Colilert-18 in subtropicalmarine and estuarine waters monitored by the Pinellas CountyHealth Department, Florida Department of Health. The highE. coli counts contrasted sharply with fecal coliform countsobtained by standard membrane filtration analysis of the samesamples. It was hypothesized that autochthonous MUG-posi-tive marine bacteria acting in tandem with ONPG-positive,MUG-negative isolates can cause false-positive E. coli resultsin the Colilert-18 test. Because previous studies indicated thatvarious plant and algal extracts can significantly interfere withthe system’s detection of both coliforms and E. coli (4), fil-

tered, cell-free marine and estuarine water samples were alsoexamined as a source of MUG and ONPG activity in theColilert-18 system.

MATERIALS AND METHODS

Enumeration of E. coli cells by Colilert-18. Marine, estuarine, and freshwatersamples were collected in sterile 100-ml glass bottles from sites located aroundTampa Bay, Fla. (Fig. 1). Eight marine, two estuarine, and four freshwater siteswere sampled. The samples were transported on ice to the laboratory within 5 hand were each inoculated into one Colilert-18 Quantitray. Undiluted freshwatersamples were assayed directly, whereas marine and estuarine samples werediluted 1:10 with sterile deionized water in accordance with the manufacturer’sinstructions. The inoculated Quantitrays were subsequently sealed and incubatedat 35.0°C for 18 to 20 h. Following incubation, the Quantitray wells were read forY color, indicating ONPG hydrolysis, and fluorescence, indicating MUG cleav-age. A handheld UV light (366 nm) was used to identify F wells. The number andtypes of well reactions in each Quantitray were translated into MPN estimatesfor E. coli according to the manufacturer’s instructions. One 100-ml sample fromeach of the eight marine and two estuarine sites was collected on 11 July, 25 July,and 29 August 2000 (see Table 2). On 11 October 2000, three 100-ml sampleswere collected at each marine site and processed as follows: one by Colilert-18 ata 1:10 (sample-deionized water) dilution, one by Colilert-18 at a 1:20 dilution,and one by the membrane filtration test for fecal coliforms. One 100-ml samplefrom each of the four freshwater sites was collected on 29 August and 11 October2000. Freshwater samples were not collected on 11 July 2000, and only onefreshwater site was sampled on 25 July 2000.

Recovery of fecal coliforms. Following incubation, the backing material of eachQuantitray was disinfected by application of 70% ethanol with a sterile swab.After the residual ethanol evaporated, sterile pipette tips were used to pierce thebacking material of two MUG-positive, ONPG-positive wells; two MUG-posi-

FIG. 1. Map of sample sites for the study. The numbered sites are marine (1 to 6, 9, and 10) or estuarine (7 and 8) water. The lettered sites(A to D) are freshwater.

540 PISCIOTTA ET AL. APPL. ENVIRON. MICROBIOL.

2 / 6 Item No. 16 Presentation Attachment 2 December 8-9, 2016

2. Colilert overestimates marine coliform bacteria

tive, ONPG-negative wells; and one MUG-negative, ONPG-positive well pertray. One tray was processed per water sample.

One hundred microliters of fluid was withdrawn from each well and added toa separate tube containing 5 ml of EC broth (Difco) and to a Durham tube. Thesamples from the Colilert wells, an E. coli-positive control, and an uninoculatedcontrol were incubated at 44.5°C in a water bath. After 24 h, all of the tubes wereexamined for turbidity and the Durham tubes were examined for gas.

At the same time the EC tubes were inoculated, fluid from each well was usedto inoculate selective-differential media. One drop (approximately 20 �l) of wellcontent was streaked for isolation on MacConkey agar and Trypticase soy agar(TSA) amended with 10 mg of MUG/liter (TSA plus MUG). Following incuba-tion at 35.0°C for 24 h, colonies were examined for lactose utilization on Mac-Conkey agar and for MUG activity on TSA plus MUG. Selected colonies isolatedfrom each Colilert well on TSA plus MUG were cross-checked on MacConkeyagar in order to assess their phenotypes with respect to lactose fermentation andon thiocitrate bile salts (TCBS) agar to determine if they belonged to the familyVibrionaceae. The same colonies were also inoculated into Colilert medium inorder to confirm their phenotypes with respect to ONPG and MUG cleavage inthe Colilert system.

Identification of environmental isolates from Colilert wells. Selected coloniesisolated from Colilert wells were identified to the genus and/or species level usingthe API 20E biochemical test system (BioMerieux, Inc., Hazelwood, Mo.). Twen-ty-four-hour-old colonies were transferred from TSA-plus-MUG plates to sep-arate 5-ml tubes containing a sterile solution of 0.85% NaCl and were resus-pended and transferred to API 20E strips. The API 20E strips were prepared,incubated, treated, and interpreted according to the manufacturer’s instructions.The numerical profile was compared with corresponding Analytab Products(API) codes in order to determine genus and species.

Examination of cell-free MUG activity. Colilert wells inoculated from sixdifferent marine sites were tested for supernatant-mediated MUG activity. Fluid(200 �l) from MUG-positive, ONPG-negative and MUG-positive, ONPG-posi-tive wells was aseptically recovered from strongly F, 24-h-old Colilert-18 wells.The fluid was filtered through a syringe filter (0.45-�m pore size) to remove cells.Twenty microliters of the filtrate from each sample was transferred to a well ofa microtiter plate containing 180 �l of Luria-Bertani broth with 10 mg of MUG/liter. Twenty microliters of the resulting 10�1 dilution was carried through adilution series to yield 10�1 to 10�6 dilutions of each sample in Luria-Bertanibroth plus MUG. The microtiter plates were examined immediately under a UVtransilluminator for fluorescence and were then incubated at 35.0°C for 72 h. Themicrotiter wells were examined for fluorescence and turbidity daily.

Marine coculture. Marine isolates with specific phenotypes (one MUG posi-tive, ONPG negative and one MUG negative, ONPG positive) were used to testthe hypothesis that two species growing in a coculture could produce a reactionin the Colilert system that would mimic E. coli. A MUG-positive, ONPG-nega-tive marine isolate was recovered from an F Colilert-18 well on TSA plus MUG.The isolate showed no growth on MacConkey agar or TCBS agar, indicating thatit was neither a coliform nor a member of the family Vibrionaceae. The API 20Eidentification indicated closest resemblance to a member of the genus Providen-cia. Vibrio cholerae O/1 was used as the ONPG-positive, MUG-negative isolate.Streaking on TSA plus MUG showed V. cholerae O/1 to be negative for MUGactivity, while API 20E analysis verified ONPG activity.

Demonstration of false-positive results from cocultures of marine bacteria.The two marine isolates described above were inoculated on separate TSA platesand incubated at 35.0°C for 48 h. Sterile swabs were used to transfer culturesfrom TSA into correspondingly labeled tubes of filtered, autoclaved seawater.Cell concentrations were normalized to 0.50 absorbance unit using a spectro-photometer set at 660 nm. A 1:1 (MUG-positive, ONPG-positive) coculture was

prepared by adding 1 ml of the suspension containing the MUG-positive isolateand 1 ml of the suspension containing the ONPG-positive isolate to 8 ml ofsterile seawater (diluted coculture). A 10-fold dilution series of the dilutedcoculture (10�2 to 10�7) was prepared in sterile seawater. Finally, 1-ml aliquotsfrom each of the 10�3 through 10�7 coculture dilutions were distributed tobottles containing 99 ml of undiluted seawater or a 1:5, 1:10, or 1:20 seawater-deionized water dilution. This was done in order to evaluate the relationshipbetween salinity levels and Colilert-18 assay results while maintaining uniforminitial inoculum concentrations and MUG-positive–ONPG-positive isolate ra-tios. Thus, a final coculture dilution series of 10�5, 10�6, 10�7, 10�8, and 10�9

was prepared for each salinity level.Culturable counts for the coculture dilution series were obtained immediately

by spread plating 100 �l from each dilution on TSA and incubating it for 24 h at35.0°C. The contents of one Colilert-18 defined-nutrient packet was then addedto each sample bottle, and the samples were transferred to Quantitrays, whichwere incubated for 18 h at 35.0°C. The reactions were then read as specifiedabove.

As a positive control for the coculture experiment, E. coli was cultured in aparallel manner, i.e., four 1-ml aliquots from the 10�6 dilution prepared in sterileseawater were inoculated into undiluted seawater and the three seawater dilu-tions specified above. E. coli cells were immediately enumerated on TSA plates,and Quantitrays were prepared and incubated as for the experimental samples.A Quantitray containing autoclaved seawater was incubated in order to verify thelack of cell-free ONPG and MUG activity. A plate count on TSA was also carriedout to verify sterility.

Fecal coliform counts on water samples were obtained by filtering 100 ml ofsample through a membrane filter (0.45-�m pore size). The filters were placedon mFC agar (Difco) and incubated for 20 to 24 h in a water bath at 44.5°C.

RESULTS

Recovery of bacteria. Ten marine and estuarine sites locatedin Tampa Bay and the Gulf of Mexico (Fig. 1) and four fresh-water sites were sampled for this study. One Colilert-18 Quan-titray was prepared for each water sample, and several wellsper Quantitray with various reactions were assessed for thepresence of fecal coliforms (Table 1).

Attempts to culture fecal coliforms from Colilert-18 wellsinoculated with marine water samples were generally unsuc-cessful, even when those wells showed the yellow fluorescent(YF) reaction characteristic of E. coli. Only 25% (15 of 60) ofEC broth tubes inoculated with fluid from YF (ONPG-posi-tive, MUG-positive) Colilert wells showed turbidity and gas(Table 1). MUG-positve, lactose-positive bacteria were iso-lated from 17 of the YF wells, including all of the EC broth-positive wells. MUG-positive, lactose-negative colonies, butnot lactose-positive, MUG-negative colonies, were readily iso-lated from YF wells that were negative by EC broth. No ECbroth-positive tubes were observed for F (MUG-positive only)wells, and only one EC broth tube inoculated from a Y wellwas positive. Isolates from this culture were, as expected, lac-tose positive and MUG negative. Over 75% of all isolates from

TABLE 1. Characterization of bacteria in individual Colilert wells by thermotolerance, lactose fermentation, and �-D-glucuronidase activity

Site Wellreaction na

No. of isolates

Growth plus gas(EC at 44.5°C)

No growth onTSA plus MUG

Lactose�

MUG�Lactose�

MUG�Lactose�

MUG�Lactose�

MUG�

Marine water YF 60 15 0 17 0 38 5Marine water F 38 0 3 0 0 26 9Marine water Y 28 1 0 0 1 11 16Freshwater YF 18 18 0 18 0 0 0Freshwater F 0 0 0 0 0 0 0Freshwater Y 8 1 0 0 2 1 5

a Number of Colilert wells sampled. The number of isolates in columns 5 to 9 add up to n (column 3).

VOL. 68, 2002 MARINE BACTERIA CAUSE FALSE POSITIVES IN COLILERT-18 541



marine sites grew on TCBS agar, indicating the presence ofVibrio spp. and other members of the family Vibrionaceae.

On no date were more than 50% of YF Colilert wells frommarine sites positive for fecal coliforms by EC broth assay, andon two sample dates all wells were negative by EC broth assay(Table 2).

Results from the four freshwater sites contrasted sharplywith those from the marine and estuarine sites (Tables 1 and2). One hundred percent (n � 18) of YF Quantitray wellssampled from freshwater sites yielded turbidity and gas in ECbroth. Lactose-positive, MUG-positive bacteria were culturedfrom all of the YF Colilert-18 wells sampled (Table 1).

Biochemical analysis of recovered isolates. In order to iden-tify organisms that could be responsible for MUG-positive (F)reactions in Colilert wells, biochemical analysis of isolates re-covered on TSA plus MUG from wells showing fluorescence

(YF and F) was carried out. When MUG-positive isolates fromfour different marine sites that were positive for fecal coliforms(growth and gas in EC broth at 44.5°C) were further charac-terized, three were identified as E. coli and one isolate did notcorrespond to a known profile. Vibrio alginolyticus and Photo-bacterium damselae were recovered from YF wells and from Fwells. All V. alginolyticus and P. damselae isolates were ONPGnegative, indicating that an unrecovered organism was respon-sible for ONPG activity in YF wells.

Some lactose-positive isolates originating from Colilert wellsinoculated with marine water were also characterized. Lactose-positive, MUG-negative colonies isolated from Y wells on TSAplus MUG were identified as Burkholderia cepacia, althoughthe certainty of identification was weak. V. cholerae was iden-tified by biochemical characterization (unconfirmed by serol-ogy or molecular techniques) in three Y wells.

From freshwater sites, biochemical characterization indi-cated that 72.7% of MUG-positive, lactose-positive isolatestested (n � 11) from YF wells were E. coli. One isolate wasidentified as Salmonella enterica serovar Arizonae, and twoisolates did not correspond to any listed API profile.

Examination of cell-free MUG activity. In order to assesswhether extracellular agents, such as enzymes or chemicals, inseawater samples had caused development of MUG-positivereactions, analysis of cell-free filtrate from YF and F Colilertwells was carried out. Uninoculated controls showed no fluo-rescence or turbidity over the course of incubation. Cell-freefiltrate of F wells from marine samples showed no develop-ment of turbidity or fluorescence at any time or at any dilutionlevel. Filtrate from one YF well developed slight fluorescencebut no yellowing in the 10�1 and 10�2 dilutions after 24 h. Thefluorescence did not appreciably increase or extend across theseries with time. Turbidity, which would have indicated bacte-rial contamination, did not develop in any well.

False-positive results caused by marine isolates. Colilert-18Quantitrays inoculated with a marine coculture (V. cholerae[ONPG positive] and Providencia sp. [MUG positive]) demon-strated the YF reactions characteristic of E. coli (Table 3).Colilert tests for each salinity level were inoculated with iden-tical coculture preparations, but inoculum plate counts differedover approximately a twofold range, depending upon the sa-linity of the medium into which the organisms were incubated(Table 3). Positive controls inoculated with �50 CFU of E. coli

TABLE 2. Percentage of YF Colilert-18 wells from which fecalcoliforms were cultured

Sitea

Percentage of wells from which fecal coliformswere culturedb

7/11/00 7/25/00 8/29/00 10/11/00

Marine and estuarine1 0 100 0 02 0 50 0 03 0 100 50 04 0 0 0 05 0 50 0 06 0 100 100 07 0 0 0 08 ND 50 0 09 0 0 50 010 0 0 0 0Total (avg) 0 45 20 0n 18 20 20 18

FreshwaterA ND 100 100 100B ND ND 100 100C ND ND 100 100D ND ND 100 100Total (avg) 100 100 100n 2 8 8

a The total number of wells sampled on each date (usually two wells per site)is denoted by n.

b ND, not determined. Dates are given as month/day/year.

TABLE 3. Inoculum plate counts and numbers of positive (YF) reactions for a marine coculture inoculated in the Colilert-18 system atvarious inoculum concentrations and in several seawater dilutions

Inoculumdilution

Result for seawater at indicated dilution

Undiluted 1:5 1:10 1:20

Inoculumplate count

(CFU ml�1)a

Positive(YF) wellsb Inoculum

plate count(CFU ml�1)

Positive(YF) wells

Inoculumplate count

(CFU ml�1)

Positive(YF) wells

Inoculumplate count

(CFU ml�1)

Positive(YF) wells

L S L S L S L S

10�5 TNTC 49 48 TNTC 49 48 TNTC 49 48 TNTC 49 4810�6 312 49 48 349 49 48 297 49 48 171 0 010�7 29 49 48 45 49 46 33 49 18 24 0 010�8 4 49 48 7 48 9 5 48 43 2 0 110�9 2 46 0 0 38 1 1 0 0 0 0 0

a TNTC, too numerous to count.b Number of YF Colilert wells per test. L, large wells (1.6 ml); S, small wells (120 �l).




ml�1 developed the expected YF reaction in all wells and ateach salinity level. No fluorescence or yellowing was seen in theuninoculated sterile-seawater control.

The intensity of coculture-mediated fluorescence was bright-est, and yellowing was weakest, in Quantitrays containing un-diluted seawater. At the 1:10 salinity level recommended bythe manufacturer, false-positive E. coli results (YF) were notedin all wells except those receiving the most dilute inoculum (0to 2 CFU ml�1, corresponding to 0 to 3 CFU in each 1.6-mlwell) (Table 3). High false-positive E. coli counts were ob-tained from Colilert tests inoculated with less than 50 CFU ofcoculture cells ml�1. For example, wells containing 1:10-di-luted seawater that were inoculated with 33 CFU of coculturecells ml�1 (10�7 dilution) were scored YF in 49 large and 18small wells, corresponding to an E. coli MPN of 307.6 CFU·100ml�1. Wells containing 1:10-diluted seawater inoculated with297 CFU of coculture cells ml�1 (10�6 dilution) were scoredYF in 49 large and 48 small YF wells, representing an E. coliMPN of �2,419 CFU·100 ml�1.

Coculture-mediated false positives dropped dramatically forthe 1:20 seawater dilution (Table 3). An inoculum concentra-tion of several thousand CFU of coculture cells per milliliter(10�5 dilution) gave false positives in all wells, but severalhundred CFU of coculture cells per milliliter (10�6 dilution)caused no false-positive results.

Unamended water samples from the marine and estuarinesites were examined concurrently by membrane filtration forfecal coliforms and by Colilert-18 tests prepared at the recom-mended 1:10 dilution for E. coli. Colilert-18 estimates of E. colinumbers were much higher at 8 of the 10 sites than corre-sponding membrane filtration estimates of fecal coliform num-bers (Table 4). However, the same samples examined at a 1:20dilution yielded Colilert-18 results that were much more con-sistent with membrane filtration results.

DISCUSSION

As coastal communities continue to expand, rapid, accuratequantitation of fecal contaminants in nearshore marine waterswill continue to be a pressing public health and environmentalconcern. While many regulatory agencies employ membranefiltration or multiple-tube fermentation assays to detect andquantify indicator bacteria, these methods are time- and labor-intensive. Consequently, assays based on disposable rapid-de-velopment kits, such as the Colilert-18 test, have been increas-ingly employed in recent years as monitoring and researchtools. Because false-positive results for E. coli can have eco-nomically harmful consequences, such as unnecessary beach

closures, it is essential that test systems yield low percentagesof false-positive and false-negative results.

This study investigated the cause of overestimates of E. colinumbers in subtropical marine waters in the United States. Adisparity in numbers estimated by Colilert-18 (E. coli) versusmembrane filtration (fecal coliforms) could not be readily ex-plained, as Colilert-18 estimates were routinely 1 to 2 orders ofmagnitude higher than estimates by membrane filtration. Thediscrepancy occurred only for marine samples and was notapparent for freshwater samples. This study showed that fecalcoliforms could not, in most cases, be cultured from the YFwells that indicate E. coli when Colilert-18 wells were inocu-lated with marine samples. Fecal coliforms, most of which wereidentified as E. coli, were recovered from all YF wells that hadbeen inoculated with freshwater samples. Control Colilert-18wells inoculated with low numbers of E. coli in seawater con-sistently yielded E. coli cultures after identical incubationtimes, indicating that the organism remained culturable underthe assay conditions.

Cell-free filtrate from YF and F marine wells did not pro-duce appreciable fluorescence or yellowing in microwells con-taining MUG, indicating that extracellular enzymes, plant ex-tracts, or free chemical agents were not the cause of thereactions. MUG-positive, ONPG-negative marine bacteriathat could have caused the development of fluorescence, in-cluding V. alginolyticus and P. damselae, were readily recoveredfrom YF and F wells.

Only one lactose-positive, MUG-negative isolate was cul-tured from the marine samples, although more were expectedgiven the high rate of false-positive YF wells. This phenome-non may be due to the difference between the substrate used inthe Colilert test (ONPG) and the substrate in MacConkey agar(lactose), from which lactose-positive colonies were recovered.Vibrio vulnificus has previously been implicated as a source of�-galactosidase activity in marine waters (5), as have otherVibrio spp. and Aeromonas spp. (13).

A coculture of two marine isolates originally isolated fromColilert-18 wells demonstrated that false-positive YF (MUG-positive, ONPG-positive) reactions can occur when the over-lapping biochemical activities of two organisms gave rise to theY color and fluorescence produced by E. coli. Cocultures in-oculated into a 1:20 seawater dilution produced false positivesonly at the highest inoculum level (approximately 3 � 103 CFUml�1), and the fluorescence was extremely faded. Unamendedmarine water samples diluted 1:10 and processed by Colilertgreatly overestimated E. coli numbers, while results for the1:20 dilution were comparable to those obtained for fecal co-liforms by membrane filtration. Although it could be argued

TABLE 4. Colilert-18 results for E. coli at marine sites in 1:10- and 1:20-diluted samples compared with membrane filtration resultsfor fecal coliforms

TestaEstimated E. coli (Colilert) or fecal coliform (MF) no. (CFU·100 ml�1)

1b 2 3 4 5 6 7 8 9 10

1:10 Colilert 143 132 7,270 373 437 130 20 10 1,467 1071:20 Colilert 20 20 20 20 20 20 20 20 40 20MF 20 20 120 20 20 20 20 20 20 20

a MF, membrane filtration.b Site number.

VOL. 68, 2002 MARINE BACTERIA CAUSE FALSE POSITIVES IN COLILERT-18 543



that the broth medium of Colilert is more favorable for recov-ery of E. coli than membrane filtration, thus consistently yield-ing higher estimates of E. coli numbers than the membranefiltration estimates of fecal coliform numbers, the results fromthe unamended marine water samples do not support thishypothesis. When E. coli concentrations estimated by Coli-lert-18 from the 1:10- and 1:20-diluted samples were com-pared, a greater-than-10-fold difference was generally ob-served (Table 4) rather than the expected 2-fold difference.The results of this work support the hypothesis that bacteriaother than fecal coliforms caused the YF reactions at the 1:10dilution. These bacteria may have been incapable of growth atthe low salinity of the 1:10 dilution, or they may have beendiluted out if they were originally present in low numbers.

While this work suggests that a 1:20 dilution of marinesamples may increase the accuracy of the Colilert-18 system forestimating E. coli numbers in tropical waters, it is important tonote that the diminution of false positives at 1:20 dilution wasdemonstrated for one pair of experimental organisms and inone set of unamended marine water samples. As there aredoubtless other species capable of causing false-positive resultsin the Colilert-18 system, the effect of sample dilution shouldbe further explored before this type of defined-substrate test isused for water quality testing in marine and estuarine waters.Furthermore, if this system is to be used in marine waters,confirmation of positive E. coli results in a medium such asEC-MUG incubated at 44.5°C would prevent most false posi-tives caused by marine bacteria.

ACKNOWLEDGMENTS

Funding for this study was provided in part by the Pinellas CountyHealth Department, Florida Department of Health.

We thank Joseph Sowers of Pinellas County Health Department,Florida Department of Health, for the map.

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3. False-positive coliform numbers using Colilert "Landre Gavriel and Lamb on Colilert.pdf



