Standard Methods: 9221A,B,C,F: Coliform bacteria by Multiple-tube test using lauryl-tryptose and EC-MUG broth

Summary
Analytes
Revisions
Data and Sites

Official Method Name

Multiple-tube Fermentation Technique for Members of the Coliform Group: 9221A. Introduction. 9221B. Standard Total Coliform Fermentation Technique 9221C. Estimation of Bacterial Density 9221F.

Current Revision

Standard Methods Online

Media

WATER

Instrumentation

Most Probable Number

Method Subcategory

Microbiological

Method Source

Standard Methods

Citation

Standard Methods Online - Standard Methods for the Examination of Water and Wastewater

Brief Method Summary

Method is especially useful when high sediment concentrations preclude membrane filtration. A dilution series of the sample is added to tubes of lauryl-tryptose broth and incubated at 35 +-0.5C for 24-48 h. The presence of growth (turbidity), an acidic reaction (yellow color) and gas is presumptive positive for total coliforms. Calculate the most-probable number (MPN) value (Standard Methods 20th ed. Section 9221C). Transfer growth from presumptive positive tubes to EC-MUG broth (E. coli broth, supplemented with the enzyme substrate 4-methylumbelliferyl-beta-D-glucuronide; Standard Methods 20th ed. Section 9221F). Incubate at 44.5+-0.2C for 24 h. Enzymatic hydrolyis of MUG is positive for E. coli, indicated by the presence of a bright-blue fluorescence using a long-wavelength ultraviolet lamp. Requirements: Ingredients for lauryl-tryptose broth and EC-MUG medium; fermentation test tubes; water baths at 35 and 44.5C; refrigeration; autoclave; a long-wave ultraviolet light source (366 nm) to read results; sterile swabs, pipets. 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, sediment, high sediment waters (especially useful when high sediment concentrations preclude membrane filtration). EPA Fed Reg (Aug 2001) for E coli, ambient only: 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. 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. 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

Applicable Concentration Range

2 to 1600 MPN/100 mL for ambient waters; 1 to 23 MPN/100 mL for finished drinking water; >1 for Presence/Absence in finished drinking water

Interferences

Substances that cause clumping of bacterial cells.

Quality Control Requirements

(Standard Methods 20th ed. Section 9020 B.8 and 9) 1. Control cultures--a positive (E. coli) for total coliforms and E. coli and negative for total coliforms (Staphylococcus) or E. coli (Enterobacter) may be used to test the medium. 2. Duplicate analyses--Perform duplicate analyses on 10% of samples. 3. Sterility check--maintain an uninoculated tube with each batch of samples.

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: Standard Methods for the Examination of Water and Wastewater, 20th Edition. L. Clesceri, A. Greenberg, and A. Eaton (editors). APHA: Washington, DC. 1998. Sections 9221B, C, and F, 9020B8 and B9, 9060B, 9221B and C, 9221F. 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.
Sample processing time 2 h. This method is labor intensive and requires a lot of incubator space. A 5 tube, 3 dilution is series recommended. Precision is improved when the results from several samples from the same sampling event are processed, estimated separately, and then mathematically combined using the geometric mean (US EPA Fed. Reg. Aug. 2001)

Maximum Holding Time

Sample should be analyzed within 6 h 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) [Drinking water can be 30 h]

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	2.000 MPN/100mL	N/A	10.00	14	14	2.00 MPN/100mL
Total coliforms(E761700) Total coliform Total coliforms	2.000 MPN/100mL	N/A	10.00	14	14	2.00 MPN/100mL

Precision Descriptor Notes:	Precision: 95 percent confidence limits (upper and lower) inferred from statistical properties of the Poisson distribution: from 1 mPN/100 mL (lower) to 10 MPN/100 mL (upper) at 2 MPN/100 mL Confidence limits increase in magnitude with increasing MPN/100 mL: see tables 9221. (II, III, IV). Accuracy: The distribution of positives in the dilution series should follow the pattern for a Poisson distribution, given in the MPN tables 9221(II, III, IV). If not, the results are suspect, and should be checked by repeating the analysis and running additional dilutions for high-level samples, or an increased number of replicates for low-level samples. General comments on precision of MPN methods (USEPA Fed Reg Aug. 2001): Unless a large number of tubes are used (five tubes per dilution/volume or more), the precision of [MPN] tests can be very poor.
Detection Level Note:	Defined by the lowest, statistically possible MPN/100 mL, when all replicates are negative. Depends on the volumes innoculated into test tubes. A detection limit of <2 MPN/100 mL corresponds to the general case for analysis of ambient waters, when five tubes each are used for dilutions of 10, 1.0, and 0.1 mL, and all results are negative.

Precision Descriptor Notes:

Precision: 95 percent confidence limits (upper and lower) inferred from statistical properties of the Poisson distribution: from 1 mPN/100 mL (lower) to 10 MPN/100 mL (upper) at 2 MPN/100 mL Confidence limits increase in magnitude with increasing MPN/100 mL: see tables 9221. (II, III, IV). Accuracy: The distribution of positives in the dilution series should follow the pattern for a Poisson distribution, given in the MPN tables 9221(II, III, IV). If not, the results are suspect, and should be checked by repeating the analysis and running additional dilutions for high-level samples, or an increased number of replicates for low-level samples. General comments on precision of MPN methods (USEPA Fed Reg Aug. 2001): Unless a large number of tubes are used (five tubes per dilution/volume or more), the precision of [MPN] tests can be very poor.

Detection Level Note:

Defined by the lowest, statistically possible MPN/100 mL, when all replicates are negative. Depends on the volumes innoculated into test tubes. A detection limit of <2 MPN/100 mL corresponds to the general case for analysis of ambient waters, when five tubes each are used for dilutions of 10, 1.0, and 0.1 mL, and all results are negative.

Revision	PDF/Link
Standard Methods Online
Standard Methods 18th, 19th, 20th ed.
Standard Methods 19th Edition (1995)
Standard Methods 18th Edition (1992)

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Standard Methods: 9221A,B,C,F: Coliform bacteria by Multiple-tube test using lauryl-tryptose and EC-MUG broth

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