IDEXX: Colilert: Coliform/E. coli Enzyme substrate test; ONPG-MUG test

Summary
Analytes
Revision
Data and Sites

Official Method Name

Colilert Test Kit Procedure

Current Revision

Current instructions with product

Media

WATER

Instrumentation

Most Probable Number

Method Subcategory

Microbiological

Method Source

IDEXX

Citation

Colilert Test Kit Procedure

Brief Method Summary

Presence/Absence: Add one packet of powder to the 100 ml water sample in a sterile, non-fluorescing vessel. Cap and shake the vessel to dissolve the powder. Incubate at 35^oC for 24 h. Read the results at 24 h (before 28 h) and compare against the comparator. If no yellow color, the test is negative, If the sample has a yellow color equal to or greater than the comparator, the presence of total coliforms is confirmed. If yellow, check for blue fluorescence by placing a 6W, 365 nm UV light within 5 inches of the sample. If blue fluorescence is greater or equal to the fluorescence of the comparator the presence of E. coli is confirmed. Quantification: See Standard Methods 20th ed. for MPN analysis (5,10,15 tube serial dilution) or pour the sample reagent mixture directly into a Quanti-Tray and seal according to instructions. Incubate and read the results as indicated above. Count the number of positive yellow wells and the number of yellow and blue-fluorescent wells. Determine the MPN/100ml using the table provided with the Quanti-Tray. For details, see the package insert. Requirements: Colilert kit from IDEXX. Sterile pipettes (for dilution only). Sterile unbuffered water for dilutions. Incubator for 35^oC. Long wavelength (365 nm) ultraviolet lamp for fluorescence readings. Reagents (comparators) in identical sample vessels for evaluation of color and fluorescence in samples. Cost of analysis: Presence/absence approx $4.00; Quantification approx. $5.00.

Scope and Application

Ambient, compliance monitoring (Standard Method 9223B). Non-compliance: freshwater (surface & ground), drinking waters, and sewage effluent; EPA Fed Reg (Aug 2001) only for E. coli in ambient waters: fresh, marine, or estuarine surface waters; applicability must be demonstrated for other matrices. Approved for presence/absence of total coliforms and E. coli in drinking water. Approved for E. coli quantitation in ambient waters; approved for presence/absence of total coliforms and E. coli in drinking water USEPA (Aug. 30, 2001). Guidelines establishing test procedures for the analysis of pollutants; Analytical methods for biological pollutants in ambient water (Aug. 20, 2004). Fed. Reg. 66(169)45811-45829. Clean Water Act section 401. 40 CFR 136.1(c). (State certification, licenses) for compliance monitoring in programs 303(c), 304(a), and 501(a). 136.3 Identification of test procedures. Pending approval for Wastewater FR, Tues., Aug. 16, 2005.

Applicable Concentration Range

For samples in wells (excluding dilution factors): 1 to 2419 MPN/100 mL for 97-well Quanti-Tray; 1 to 200 MPN/100 mL for 51-well Quanti-Tray

Interferences

Heterotrophs at greater than 2 million/100ml. Excess chlorine: blue flash seen when Colilert mixed with sample. Avoid prolonged exposure to sunlight. If a water has some color (humic, fulvic acids), evaluate the color of the sample with Colilert by comparison to a control without Colilert.

Quality Control Requirements

Control cultures: conducted on each lot of Colilert received as a minimum. Inoculate sterile water with either ATCC bacteria or Quanti-Cult (IDEXX)- E.coli (color & fluorescent), K. Pneumoniae (color only) and P. aeruginosa (No color or fluorescence).

Sample Handling

Sample preservation: chilled, 1^oC to 4^oC; 0.0008% (w/w) Na2S2O3 added to chlorinated waters EPA Fed Reg (Aug 2001). Techniques for collection: Standard Methods for the Examination of Water and Wastewater, 20th Edition. L. Clesceri, A. Greenberg, and A. Eaton (editors). APHA: Washington, DC. 1998. Sections 9020B8 and B9, 9060A and B.
Sample processing time: less than 5 minutes. Dilute marine samples 1:10 with sterile fresh water.

Maximum Holding Time

Sample should be analyzed within 6 h after sampling and within 2 h from receipt of sample in lab for compliance or 24 h for routine monitoring (Standard Methods, 20th ed., Section 9060B); however, a 6 h holding time for all samples is highly recommended (Myers and Sylvester, 1997).

Relative Cost

Less than $50

Sample Preparation Methods

This method has 2 analytes associated with it.

Analyte	Detection Level	Bias	Precision	Pct False Positive	Pct False Negative	Spiking Level
E. coli(68583-22-2) E. coli E.coli Escherichia coli	1.000 MPN/100mL	N/A	N/A
Total coliforms(E761700) Total coliform Total coliforms	1.000 MPN/100mL	N/A	N/A

Precision Descriptor Notes:	Covert, T.C.; Rice, E. W.; Johnson, S. A.; Berman, D.; Johnson, C. H.; Mason, P. J. 1992. Comparing defined substrate tests for the detection of E. coli in water. Jour. AWWA.): Covert, T.C.; Shadix, L. C.; Rice, E. W.; Haines, J. R.; Freyberg, R. W. 1989. Evaluation of the autoanalysis Colilert test for detection and enumeration of total coliforms. Appl. Environ. Microbiol. 55(10):2443-7. Eckner, K. 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(8):3079-3083.
Detection Level Note:	For samples in wells (excluding dilution factors), determined by a) the largest volume that can be used without dilution to be in the concentration range for analysis; b) the lowest, statistically possible MPN/100 mL, when all replicates are negative. For example, if a 100 mL sample is used without dilution, the detection limit would be < 1 MPN/100 mL.

Precision Descriptor Notes:

Covert, T.C.; Rice, E. W.; Johnson, S. A.; Berman, D.; Johnson, C. H.; Mason, P. J. 1992. Comparing defined substrate tests for the detection of E. coli in water. Jour. AWWA.): Covert, T.C.; Shadix, L. C.; Rice, E. W.; Haines, J. R.; Freyberg, R. W. 1989. Evaluation of the autoanalysis Colilert test for detection and enumeration of total coliforms. Appl. Environ. Microbiol. 55(10):2443-7. Eckner, K. 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(8):3079-3083.

Detection Level Note:

For samples in wells (excluding dilution factors), determined by a) the largest volume that can be used without dilution to be in the concentration range for analysis; b) the lowest, statistically possible MPN/100 mL, when all replicates are negative. For example, if a 100 mL sample is used without dilution, the detection limit would be < 1 MPN/100 mL.

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IDEXX: Colilert: Coliform/E. coli Enzyme substrate test; ONPG-MUG test

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