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SIMPLIFICATION OF THE KWIKCOUNT® EC MF METHOD (USEPA Approved - 40 CFR Part 136.3)

 

(Original Testing and Data by Fu-Chih Hsu and Rebecca Wong of Scientific Methods Inc)

Following are suggestions of how to simplify the KwikCount® EC MF Method by eliminating the Petri dish apparatus that was used in achieving the USEPA official approval as listed in the Federal Register. That Method requires the use of a 47mm sterile Petri Dish, containing a sterile absorbent pad, and a micropore membrane filter. Protocol for the USEPA approved method requires the preparation of the pad in the bottom of the Petri Dish by depositing 1.8 ± mL of the sterile nutrient broth from the 10 mL bottle prior to filtering the aqueous sample (size 1-100 mL) through a micropore membrane filter, and then placing the filter (right side up) on top of the broth soaked pad. This prepared dish containing the sample is then incubated at 41°±.5 C for 8 hrs, when it can be observed in a dark room with  UV long wave(366nm)  light and the fluorescing E. coli colonies can be accurately counted.

A much simpler, time saving and less expensive way of achieving equally accurate results can be obtained by using the R-CARD® KwikCount® EC, which had not been fully developed at the time that the studies by Scientific Methods Inc. used to obtain USEPA approval were completed and submitted to the USEPA. Therefore, the approval did not include this R-CARD® application.

The R-CARD® KwikCount® EC is made to contain the same formulation of nutrients as are in the 20 mL bottle of liquid that is included in the KwikCount® EC MF Method. There are at least two ways to utilize the R-CARD® that give the same results as the EPA approved method. First, the sample can be filtered with the same micropore filter and the filter containing the bacteria is then placed in the center of the R-CARD® bottom (top lifted) and 1mL of sterile buffered or deionized water is deposited on the top of the membrane. The R-CARD® top piece is then lowered onto the wet filter, which is incubated 8-10 hrs (41°±.5 C) as is the approved dish, removed and subjected to long wave UV light in a darkroom. Any E. coli will fluoresce and be easily counted, just as in the KwikCount® EC MF approved method.

Easier yet, the aqueous sample can be diluted in sterile water or sterile buffered water so that each mL contains an appropriate number of the target E. coli (recommended 20-100 CFU/mL). Then, using a sterile 1mL dropper, lift the top piece of the CARD, and deposit 1 mL of the diluted aqueous sample on the middle of the bottom piece and drop the top piece down onto it. The water will spread into a large circle and gel within 2 minutes. Incubate and count as before. It should be noted that this last approach saves time and money spent on the membrane filter and filtering, as well as the Petri dish cost.

If you wish to use any of the above described CARD methods, you must not assume that they are included in the USEPA approved Method for Ambient Water. If approval is mandatory for your use, you must discuss your proposed protocol change with you area officials to see if there may be any options agreeable (such as side by side study with the approved method, etc.) that are acceptable.

The following comparative counts, photographs, and tables contain the date done by Roth Bioscience, LLC done in sets of four, with the inoculum being E. coli ATCC 25922. All cards or MF dishes were done at the same time, incubated at 41° C, and the results were counted at the same time. It can be noted that at 8 hours, resulting counts were slightly lower than the 10 hour results, which may be interpreted that with this inoculum (E. coli 25922), 8 hours incubation should be used to indicate only the presence/absence of the target organism, with 10 hours incubation being necessary to assure the accurate total population.

EPA FEDERAL REGISTER /VOLUME 86. No. 95/ WEDNESDAY, MAY 19, 2021/ RULES AND REGULATIONS  CLICK HERE

Page 27325 I.“Changes to 49 CFR 136.3 To Include Alternate Test Procedures (ATPs)” Lists the KwikCount® EC Medium test as approved. (It also lists it as being obtained from Micrology Laboratories, LLC, 1303 Eisenhower Drive, Goshen, IN 45326.) That Corporate name was changed to Roth Bioscience, LLC as of June 3, 2019.

Page 27253 II. TABLE IH---“LIST OF APPROVED MICROBIOLOGICAL METHODS FOR AMBIENT WATER” ALSO LISTS KwikCount® EC.

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Materials and Methods

Inoculum: E.coli ATCC 25922 (a week old culture)

Test Method: R-CARD® KWIKCOUNT® EC
                       KWIKCOUNT® EC MF (USEPA APPROVED METHOD)

Preparation: A dilution was prepared using sterile water. One mL of sample was added onto R-CARD® KWIKCOUNT® EC. One mL of sample was filtered using membrane filtration method and the micropore filter (0.45µm pore - 47mm) is then placed onto the plated with KWIKCOUNT® EC MF  broth added to the pad. All samples were tested in quadruplicates (#1 - #4).

Photo of the CARD #1  and Plate #1

Both methods far superior to standard petri dishes with the outdated agar based, thick media layer or other variable methods. Results equal or superior recovery of target organisms.

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