Modified mTEC Agar

Cat. no. G106 Modified mTEC Agar, 15x60mm Plate, 11ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Modified mTEC Agar is recommended for selective chromogenic differentiation and enumeration of Escherichia coli in water, using membrane filtration.

SUMMARY

The density of Escherichia coli in water samples has proven to accurately represent the degree of pollution and therefore sanitary quality of ambient, drinking and waste water. (1) While previous methods of enumerating E. coli required the use of multiple media types and longer incubation, Dufour et al. produced a membrane filter procedure in 1981 that did not require subculture or further identification of isolates. (2) This method does however require transfer of the filter to a urea substrate for final identification.

Hardy Diagnostics Modified mTEC (membrane Thermotolerant E. coli ) Agar allows for a one step, one medium method for differentiation and enumeration of E. coli that does not require any transfer of the filter. Method 1603, published by the EPA in 2002, recommends this media as a measure of fresh, estuarine and marine water quality. (3)

Modified mTEC Agar contains peptones as a source of carbon, nitrogen, vitamins and minerals. Yeast extract supplies complex B vitamins, trace elements and amino acids that help stimulate bacterial growth. Lactose is a carbohydrate that can be fermented by E. coli at elevated temperatures. The buffers in this formula are monopotassium phosphate and dipotassium phosphate. Agar is used to solidify the media, while sodium lauryl sulfate and sodium deoxycholate serve to inhibit gram-positive bacteria. This media contains a chromogen (5-Bromo-6-Chloro-3-Indoyl-beta-D-Glucuronide) which is converted into glucuronic acid. This compound produces red or magenta colonies in the presence of E. coli strains possessing the enzyme beta-D-glucuronidase. (3)

FORMULA

Ingredients per liter of deionized water:*

Lactose 10.0gm
Sodium Chloride 7.5gm
Proteose Peptone No. 3 5.0gm
Dipotassium Phosphate 3.3gm
Yeast Extract 3.0gm
Monopotassium Phosphate 1.0gm
5-Bromo-6-Chloro-3-Indoyl-beta-D-Glucuronide 0.5gm
Sodium Lauryl Sulfate 0.2gm
Sodium Deoxycholate 0.1gm
Agar 15.0gm

Final pH 7.3 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Sample Collection:

Consult listed references for information on sample collection. (4-7) The sample collection and filtration technique are identical to those used in the original mTEC procedure. (3)

Method of Use (1) :

Follow applicable membrane filter procedures. (1)

1. Roll membrane filter used to collect the water sample onto the agar surface. Avoid the formation of air bubbles between the filter and agar surface. Run the forceps around the perimeter of the membrane to ensure it is properly seated on the agar. (3)

2. Invert plates and incubate for 2 hours at 35 +/- 2ºC. in order to revive stressed cells.

3. Transfer the plates to a sealed bag and place in a waterbath at 44.5 +/- 0.5ºC. and incubate for 22-24 hours.

4. Count and record all red or magenta colonies. Illuminated lens with two to five times magnification or a stereoscopic microscope may be used to aid in enumeration.

INTERPRETATION OF RESULTS

All red or magenta colored colonies can be verified as E. coli and may be presumptively identified. All such colonies should be counted and reported per volume of water sampled. Dilution factors must be taken into account. Further verification may be done according to Standard Methods for the Examination of Water and Wastewater or Method 1603. (1,3)

LIMITATIONS

Consult references for appropriate water sample size choice to ensure countable plates (20 to 80 colonies per filter). (1) Identification may be inaccurate if samples contain particulates suspended in the water; this material can clog the membrane and prevent filtration or cause spreading of colonies. (3) Minimize the exposure to light as it may damage the chromogen. (3)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, membrane filters, forceps, other culture media, water baths, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Escherichia coli
ATCC ® 25922
MF 20-24 hrs 44.5°C Aerobic Growth; reddish-purple or magenta colonies
Enterobacter aerogenes
ATCC ® 13048
MF 20-24 hrs 44.5°C Aerobic Inhibited

USER QUALITY CONTROL

REFERENCES

1. American Public Health Association. Standard Methods for the Examination of Water and Wastewater, APHA, Washington, D.C.

2. Dufour, A.P., et al. 1981. Membrane filter method for enumerating Escherichia coli. Appl. Environ. Microbiol .; 41(5):1152-1158.

3. United States Environmental Protection Agency. 2002. Method 1603: Escherichia coli ( E. coli ) in water by membrane filtration using modified membrane-thermotolerant Escherichia coli agar (modified mTEC). Publication EPA 821-R-02-023 Office of Water 4303T, Washington D.C.

4. Anderson, N.L., et al. 2005. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

5. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

6. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

7. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.


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