IDEXX: Colilert-18: Coliform/E. coli Enzyme substrate test; ONPG-MUG test
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Official Method Name
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Colilert-18 Test Kit Procedure |
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Current Revision
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Media
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WATER |
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Instrumentation
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Most Probable Number |
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Method Subcategory
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Microbiological |
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Method Source
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Citation
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Brief Method Summary
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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. Pre-warm sample (with Colilert-18) in a 35C water bath for 20 minutes or 44.5C water bath for 7-10 minutes. This is part of the total incubation period. Incubate at 35 degrees C for 18 h. Read the results at 18 h (before 22 h) and evaluate color 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-18 kit from IDEXX. Sterile pipets. Antifoaming reagents. Sterile, unbuffered water for dilutions. Water bath for warming samples to 33 degrees C - 45 degrees C. Incubator for 35 degrees C controlled within 0.5 degrees C. 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. |
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Scope and Application
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Ambient, compliance monitoring (Standard Method 9223B): Freshwater (surface & ground), drinking waters, sewage effluent and saltwater 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 (August 30, 2001). Guidelines establishing test procedures for the analysis of pollutants; Analytical methods for biological pollutants in ambient water (Final Ambient Guidelines 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, Tuesday, Aug 16th, 2005. |
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Applicable Concentration Range
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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 |
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Interferences
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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. |
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Quality Control Requirements
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Control cultures: Conducted on each lot of Colilert-18 received as a minimum. Inoculate sterile water with either ATCC bacteria or Quanti-Cult (IDEXX)- E. coli (color & fluorescence), K. Pneumoniae (color only) and P. aeruginosa (no color or fluorescence). |
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Sample Handling
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Sample preservation: chilled, 1oC to 4oC; 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. |
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Maximum Holding Time
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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). |
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Relative Cost
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Less than $50 |
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Sample Preparation Methods
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