EPA Method 1605

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EPA Method 1605:
Aeromonas in Finished Water by Membrane Filtration. Official Name: Method 1605: Aeromonas in Finished Water by Membrane Filtration using Ampicillin-Dextrin Agar with Vancomycin (ADA-V)

Summary:
The method provides a direct count of Aeromonas species in water based on the growth of yellow colonies on the surface of the membrane filter using a selective medium. A water sample is filtered through 0.45-um-pore-size membrane filter. The filter is placed on ampicillin-dextrin agar with vancomycin (ADA-V) and incubated at 35 degrees C +/- 0.5 degrees C for 24 +/- 2 hours. This medium uses ampicillin and vancomycin to inhibit non-Aeromonas species, while allowing most Aeromonas species to grow. The medium uses dextrin as a fermentable carbohydrate, and bromothymol blue as an indicator of acidity produced by the fermentation of dextrin. Presumptively identified yellow colonies are counted and confirmed by testing for the presence of cytochrome c (oxidase test), and the ability to ferment trehalose and produce indole. The membrane filtration procedure provides a direct count of culturable Aeromonas in water samples that is based on the growth of bacterial colonies on the surface of the membrane filter placed on a selective medium.

Scope:
This method describes a membrane filter (MF) procedure for the detection and enumeration of Aeromonas species in finished water samples. Aeromonas is a common genus of bacteria indigenous to surface waters. Its numbers are more likely to be greater during periods of warmer weather and when increased concentrations of organic nutrients are present. It is also more likely to be found in non-chlorinated water distribution systems or low-flow parts of chlorinated systems. Some Aeromonas species are opportunistic pathogens. This method was subjected to an interlaboratory validation study involving 11 laboratories and 11 finished drinking water matrices. This method was not validated for other water types. Use of this method and appropriate validation for other water types is the responsibility of the user.

Citation:
Method 1605: Aeromonas in Finished Water by Membrane Filtration Using Ampicillin-Dextrin Agar with Vancomycin (ADA-V), EPA Office of Water, 2001. EPA/821/R-01/034

Interferences:
Water samples containing colloidal or suspended particulate material may clog the membrane filter and prevent filtration or cause spreading of bacterial colonies which could interfere with identification of target colonies. Other ampicillin/vancomycin resistant bacteria that are not aeromonads may be able to grow on this medium. Some of these bacteria may also produce yellow colonies if they are able to produce acid byproducts from the fermentation of dextrin or some other media component, or if they produce a yellow pigment. Enterococcus are reported to produce pinpoint-size yellow colonies on ADA. Confirmation of presumptive Aeromonas colonies is necessary to mitigate false positives.

QC Requirements:
Specific quality control (QC) requirements for Method 1605 are provided in Section 9 of the method. Each laboratory that uses Method 1605 is required to operate a formal quality assurance (QA) program. The minimum QA requirements consist of the initial demonstration of capability (IDC) test, ongoing analysis of spiked reagent water (ODC test) and spiked finished drinking water samples (MS/MSD), and analysis of negative culture controls, dilution/rinse water blanks, and media sterility checks as tests of continued acceptable performance.

Maximum Holding Time:
Samples must be analyzed within 30 hours of sample collection.

Media:
WATER

Subcategory:
Microbiological

Precision:
This method was subjected to an interlaboratory validation study involving 11 laboratories and 11 finished drinking water matrices.

Detection:
-

Revision Number:
1-Oct