EPA Method 1604

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EPA Method 1604:
Total Coliforms and E. coli in Drinking Water by Membrane Filtration. Official Name: Method 1604: Total Coliforms and Escherichia coli in Water by Membrane Filtration Using a Simultaneous Detection Technique (MI Medium)

An appropriate volume of a water sample (100 mL for drinking water) is filtered through a 47-mm, 0.45-?m pore size cellulose ester membrane filter that retains the bacteria present in the sample. The filter is placed on a 5-mL plate of MI agar or on an absorbent pad saturated with 2-3 mL of MI broth, and the plate is incubated at 35 degrees C for up to 24 hours. The bacterial colonies that grow on the plate are inspected for the presence of blue color from the breakdown of IBDG by the E. coli enzyme B-glucuronidase and fluorescence under longwave ultraviolet light (366 nm) from the breakdown of MUGal by the TC enzyme B-galactosidase.

This test method describes a sensitive and differential membrane filter (MF) medium, using MI agar or MI broth, for the simultaneous detection and enumeration of both total coliforms (TC) and Escherichia coli (E. coli) in water samples in 24 hours or less on the basis of their specific enzyme activities. Two enzyme substrates, the fluorogen 4-Methylumbelliferyl-B-D-galactopyranoside (MUGal) and a chromogen Indoxyl-B-D-glucuronide (IBDG), are included in the medium to detect the enzymes B-galactosidase and B-glucuronidase, respectively, produced by TC and E. coli, respectively. This method will be used primarily by certified drinking water laboratories for microbial analysis of potable water. Other uses include recreational, surface or marine water, bottled water, groundwater, well water, treatment plant effluents, water from drinking water distribution lines, drinking water source water, and possibly foods, pharmaceuticals, clinical specimens (human or veterinary), other environmental samples (e.g., aerosols, soil, runoff, or sludge) and/or isolation and separation of transformants though the use of E. coli lac Z or gus A/uid reporter genes.

Method 1604: Total Coliforms and Escherichia coli in Water by Membrane Filtration Using a Simultaneous Detection Technique (MI Medium), EPA Office of Water, September 2002. EPA/821/R-02/024

Water samples containing colloidal or suspended particulate material can clog the membrane filter, thereby preventing filtration, or cause spreading of bacterial colonies which could interfere with identification of target colonies. However, the blue E. coli colonies can often be counted on plates with heavy particulates or high concentrations of total bacteria. The presence of some lateral diffusion of blue color away from the target E. coli colonies can affect enumeration and colony picking on plates with high concentrations of E. coli. This problem should not affect filters with low counts, such as those obtained with drinking water or properly diluted samples.

QC Requirements:
Pretest each batch of MI agar or broth for performance (i.e., correct enzyme reactions) with known cultures (E. coli, TC, and a non-coliform). Test new lots of membrane filters against an acceptable reference lot using the method of Brenner and Rankin. Perform specific filtration control tests each time samples are analyzed, and record the results. See recommendations on quality control for microbiological analyses in the ?Manual for the Certification of Laboratories Analyzing Drinking Water: Criteria and Procedures; Quality Assurance? and the USEPA Microbiology Methods Manual, part IV, C.

Maximum Holding Time:
Analyze samples as soon as possible after collection. Drinking water samples should be analyzed within 30 h of collection. Do not hold source water samples longer than 6 h between collection and initiation of analyses, and the analyses should be complete within 8 h of sample collection.



The single lab recovery of E. coli is reported to be 97.9% of the Heterotrophic Plate Count (pour plate) and 115% of the R2A spread plate. For Klebsiella pneumoniae and Enterobacter aerogenes, two total coliforms, the recoveries are 87.5% and 85.7% of the HPC, respectively, and 89.3% and 85.8% of the R2A spread plate, respectively. The specificities for E. coli and total coliforms are reported to be 95.7% and 93.1%, respectively. The single lab coefficients of variation for E. coli and total coliforms are reported to be 25.1% and 17.6%, respectively, for a variety of water types. In a collaborative study, 19 laboratories concurrently analyzed six wastewater-spiked Cincinnati tap water samples, containing 3 different concentrations of E. coli ( 30 per 100 mL). The single laboratory precision (coefficient of variation), a measure of the repeatability, ranged from 3.3% to 27.3% for E. coli and from 2.5% to 5.1% for TC for the six samples tested, while the overall precision (coefficient of variation), a measure of reproducibility, ranged from 8.6% to 40.5% and from 6.9% to 27.7%, respectively. These values are based on log10-transformed data.

The detection limits of this method are one E. coli and/or one total coliform per sample volume or dilution tested.

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