EPA Method 1600

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EPA Method 1600:
Enterococci in water by membrane filtration using mEI Agar. Official Name: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl-B-D-Glucoside Agar (mEI)

Summary:
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.

Scope:
This method describes a membrane filter procedure for the detection and enumeration of the enterococci bacteria in water.

Citation:
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.

Interferences:
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.

QC Requirements:
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.

Maximum Holding Time:
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.

Media:
WATER

Subcategory:
Microbiological

Precision:
-

Detection:
-

Revision Number:
6-Jul