Standard Methods: 9221E:  Fecal coliforms by Multiple tube fermentation

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Official Method Name
Multiple-tube Fermentation Technique for Members of the Coliform Group: 9221C. Estimation of Bacterial Density. 9221E Fecal Coliform Procedure In Standard Methods for the Examination of Water and Wa
Current Revision
Standard Methods Online
Most Probable Number
Method Subcategory
Method Source
  Standard Methods
  Standard Methods Online - Standard Methods for the Examination of Water and Wastewater
Brief Method Summary
Requires the addition of a dilution series of the sample to tubes of EC medium or A-1 broth. For EC medium, incubate at 44.5+-0.2C for 24 h. For A-1 broth, incubate for 3 h at 35+-0.5C and then 21 h at 44.5+-0.2C. The presence of gas production in EC medium or A-1 broth is a positive test for fecal coliforms. Calculate the most-probable number (MPN) value. Requirements: ingredients for EC medium and A-1 broth; fermentation test tubes; water baths at 35 and 44.5C; refrigeration; autoclave; sterile swabs, pipets. Cost of analysis (USEPA Fed. Reg. Aug 2001): fecal coliforms $21 ($15 to $35).
Scope and Application
ambient, compliance monitoring: all water, sediment. 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: Total Coliform Rule: presence/absence of total coliforms, fecal coliforms or E coli
Applicable Concentration Range
most probable number (MPN) /100mL
None Provided.
Quality Control Requirements
(Standard Methods, 20th Edition 9020 B.8 and 9) 1. Control cultures--positive (E. coli) and negative (Enterobacter) control cultures 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 9020B8 and B9, 9060A and B. 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. 5 tube, 3 dilution 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 Edition 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