EPA-OW: 1600: Enterococci in water by membrane filtration using mEI Agar
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Official Method Name
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Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-B-D-Glucoside Agar (mEI) |
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Current Revision
| July 2006 |
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Media
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WATER |
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Instrumentation
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Membrane Filtration |
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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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USEPA, 2006, Method 1600: Enterococci in water by membrane filtration using membrane-Enterococcus Indoxyl-ß-D-Glucoside Agar (mEI): U.S. Environmental Protection Agency Report 821-R-06-009, 42 p. |
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Brief Method Summary
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This method provides a direct count of bacteria in water based on the development of colonies on the surface of the membrane filter. A water sample is filtered through the membrane which retains the bacteria. Following filtration, the membrane containing the bacterial cells is placed on a selective medium, mEI agar, and incubated for 24 hours at 41oC + 0.5oC. All colonies greater than or equal to 0.5 mm in diameter (regardless of color) with a blue halo are recorded as enterococci colonies. A fluorescent lamp with a magnifying lens is used for counting to give maximum visibility of colonies. This is a single-step method that is a modification of EPA Method 1106.1 (mE-EIA). Unlike the mE-EIA method, it does not require the transfer of the membrane filter to another medium. The modified medium has a reduced amount of triphenyltetrazolium chloride (TTC) and includes indoxyl B-D-glucoside, a chromogenic cellobiose analog used in place of esculin. In this procedure, B-glucosidase-positive enterococci produce an insoluble indigo blue complex which diffuses into the surrounding media, forming a blue halo around the colony. |
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Scope and Application
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This method describes a membrane filter procedure for the detection and enumeration of the enterococci bacteria in water. |
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Applicable Concentration Range
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Interferences
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Water samples containing colloidal or suspended particulate materials can clog the membrane filter and prevent filtration, or cause spreading of bacterial colonies which could interfere with enumeration and identification of target colonies. |
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Quality Control Requirements
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The minimum analytical QC requirements for the analysis of samples using this method include an initial demonstration of laboratory capability through performance of the initial precision and recovery (IPR) analyses, ongoing demonstration of laboratory capability through performance of the ongoing precision and recovery (OPR) analysis and matrix spike (MS) analysis (disinfected wastewater only), and the routine analysis of positive and negative controls, filter sterility checks, method blanks, and media sterility checks. For the IPR, OPR and MS analyses, it is necessary to spike samples with either laboratory-prepared spiking suspensions or BioBalls. |
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Sample Handling
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Samples are collected by hand or with a sampling device if the sampling site has difficult access such as a dock, bridge, or bank adjacent to surface water. Composite samples should not be collected, since such samples do not display the range of values found in individual samples. The sampling depth for surface water samples should be 6-12 inches below the water surface. Sample containers should be positioned such that the mouth of the container is pointed away from the sampler or sample point. After removal of the container from the water, a small portion of the sample should be discarded to allow for proper mixing before analyses. Ice or refrigerate water samples at a temperature of <10oC during transit to the laboratory. Do not freeze the samples. Use insulated containers to assure proper maintenance of storage temperature. Ensure that sample bottles are not totally immersed in water during transit or storage. |
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Maximum Holding Time
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Analysis preferably within 2 hours of collection; max. transport time to lab is 6 hours, and samples should be processed within 2 hours of receipt at lab. |
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Relative Cost
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Less than $50 |
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Sample Preparation Methods
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