Hach Co.: 10029: E. coli by m-ColiBlue24 Broth Procedure for Membrane Filtration

Summary
Analytes
Revision
Data and Sites

Official Method Name

Coliforms: Membrane Filtration (simultaneous detection) In: Hach Analytical Procedures. m-ColiBlue24 Broth Procedure for Membrane Filtration. Hach Product Literature #8433. Hach: Loveland, CO. Dec.

Current Revision

Dec-99

Media

WATER

Instrumentation

Membrane Filtration

Method Subcategory

Microbiological

Method Source

Hach Co.

Citation

m-ColiBlue24 Broth Procedure for Membrane Filtration. Hach Product Literature #8433.

Brief Method Summary

100 ml water sample is filtered through a 0.45 micron membrane filter. 2 ml of the reagent is poured onto a sterile pad in a 50 mm petri pad. The filter is then transferred to the plate, covered and incubated at 35C for 24 h. The presence of red colonies is indicative of coliforms, which reduce TTC (2,3,5-triphenyltetrazolium chloride). The presence of blue colonies is indicative of E. coli, which hydrolyze the enzyme substrate BCIG (5-bromo-4-chloro-3-indolyl-beta-D-glucuronide) to an insoluble salt. The use of a low power (10-15 magnification) binoocular wide-field dissecting microscope or equivalent may be necessary to provide optimal viewing of the colonies. Requirements: m-ColiBlue24 medium; buffer for rinsing and dilutions; sterile pipets. Culture dishes (50x10mm); 0.45 micron membrane filters; Refrigeration; autoclave; manifold and sterile filter funnel. Magnifying glass; forceps, alcohol; incubator at 35+/-0.5C. A low power (10-15 magnification) binocular wide-field dissecting microscope or equivalent may be necessary to provide optimal viewing of the colonies. Cost of analysis (USEPA Fed. Reg. Aug 2001): E. coli $22 ($10 to $35) total coliforms $22 ($15 to $48).

Scope and Application

ambient, compliance monitoring. non-compliance: all water - surface water, ground water, drinking water, bottled water, recreational water, wastewater EPA Fed Reg (Aug 2001) for E. coli, ambient only: fresh, marine, or estuarine surface waters; applicability must be demonstrated for other matrices. Hach method 10029 approved for drinking water by US EPA Jan. 3, 2000. USEPA. 1999 (December 1). National primary and secondary drinking water regulations:analytical methods for chemical and microbiological contaminants and revisions to laboratory certification requirements; final rule. Fed. Reg. 64(230)67449-67467. Safe Drinking Water Act: a) Total Coliform Rule: presence/absence of total coliforms and E. coli b) Surface Water Treatment Rule: enumeration of total coliforms. USEPA. 2001 (August 30). Guidelines establishing test procedures for the analysis of pollutants; Analytical methods for biological pollutants in ambient water; proposed rule. 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.

Applicable Concentration Range

20 - 80 CFU/100 mL is considered ideal for enumeration. Maximum: 200 CFU/100 mL; dilution is required for samples that exceed this level.

Interferences

The commercial product contains a nutritive lactose-based medium with inhibitors to eliminate growth of non-coliforms. Waters containing sediment or suspended materials such as iron and manganese can clog the filter pores and prevent filtration or mask target colonies and prevent accurate counting. Toxic materials such as meals, caustics, chloramines may also adversely affect the recovery of E. coli.

Quality Control Requirements

Testing should be conducted on every lot of reagent with a known positive and negative strain. Verify filters are free of coliform bacteria.

Sample Handling

Sample preservation: chilled, 1 to 4 C; 0.0008% (w/w) Na2S2O3 added to chlorinated waters EPA Fed Reg (Aug 2001). Techniques for collection: Myers, D.N.; Sylvester, F.D. 1997. National field manual for the collection of water-quality data - biological indicators. USGS Techniques of Water Resources Investigations. Book 9, Chapter A7. 38 pp. 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, 9222B.
Sample processing time: greater than 10 minutes. Recommended sample volumes to be diluted to 100 mL and filtered, according to water type, are (Standard Methods, 20th ed. Table 9222:III): 100 mL lakes, reservoirs, wells, springs; 50 mL drinking water sources, natural bathing waters; 1 - 10 mL, sewage treatment; 0.1 - 1 mL, farm ponds and streams, stormwater runoff; 0.01 - 0.1 mL, raw municipal sewage and feedlot runoff; 0.001 - 0.01 mL sewage sludge. To minimize interferences causing underestimation of organism density in MF methods (USEPA Fed Reg Aug. 2001): replicates of smaller sample dilutions/volumes may be filtered and the results combined.

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, for all samples, holding less than 6 h 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 CFU/100 mL	N/A	N/A	3	0
Total coliforms(E761700) Total coliform Total coliforms	1.000 CFU/100 mL	N/A	N/A	27	1

Precision Descriptor Notes:	Accuracy: Total Coliforms: sensitivity 98.9%, specificity 78.7% false positive error 26.8%, false negative error 1.1%. E. coli: sensitivity 100.0%, specificity 97.7% false positive error 2.5%, false negative error 0%. Grant, M.A. 1997. A new membrane filtration medium for simultaneous detection and enumeration of Escherichia coli and total coliforms. Appl. Environ. Microbiol. 63(9):3526-3530. General comments (USEPA Fed Reg Aug. 2001): Membrane filter (MF) methods are generally more precise in enumerating target organisms than MPN tests. Based on susceptibility to interferences, MF tests may underestimate the number of viable bacteria.
Detection Level Note:	Determined by using the largest volume plated, and the lowest number of organisms that can be counted. For example, if 100 mL was the largest volume plated, the detection would be 1 colony per 100 mL.

Precision Descriptor Notes:

Accuracy:

Total Coliforms: sensitivity 98.9%, specificity 78.7% false positive error 26.8%, false negative error 1.1%.
E. coli: sensitivity 100.0%, specificity 97.7% false positive error 2.5%, false negative error 0%.
Grant, M.A. 1997. A new membrane filtration medium for simultaneous detection and enumeration of Escherichia coli and total coliforms. Appl. Environ. Microbiol. 63(9):3526-3530.
General comments (USEPA Fed Reg Aug. 2001): Membrane filter (MF) methods are generally more precise in enumerating target organisms than MPN tests. Based on susceptibility to interferences, MF tests may underestimate the number of viable bacteria.

Detection Level Note:

Determined by using the largest volume plated, and the lowest number of organisms that can be counted. For example, if 100 mL was the largest volume plated, the detection would be 1 colony per 100 mL.

Revision	PDF/Link
Dec-99

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Hach Co.: 10029: E. coli by m-ColiBlue24 Broth Procedure for Membrane Filtration

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