NUTRIENT AGAR with MUG

Cat. no. G114 Nutrient Agar with MUG, 15x60mm Plate, 11ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Nutrient Agar with MUG is recommended for detecting and enumerating Escherichia coli in water samples.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Escherichia coli is a member of the fecal coliform group of bacteria and its presence in water samples is indicative of fecal contamination.(1) Feng and Hartman developed a rapid assay for E. coli by incorporating 4-methylumbelliferyl-β-D-glucuronide (MUG) at a final concentration of 100µg/ml into Lauryl Tryptose Broth.(2) Nutrient Agar is modified the same way with the addition of MUG. Rapid quantitation and verification may be achieved with the membrane filtration method by transferring the membrane from a total coliform or fecal coliform positive sample to a Nutrient Agar substrate containing MUG.(1) Mates and Shaffer used the membrane filter-Endo LES Agar method, followed by incubation on Nutrient Agar with MUG, to detect and enumerate E. coli within 4 hours of membrane transfer.(3) E. coli was recovered at a rate of 98% with no false-positive results.

The formulation of Nutrient Agar is composed of peptone, beef extract and agar. Nutrients necessary for the replication and growth of a large number of nonfastidious microorganisms are provided by the simple formulation. Water soluble substances including carbohydrates, vitamins, organic nitrogen compounds and salts are present in the beef extract.(10) Peptone supplies the principle source of organic nitrogen in the form of amino acids and long-chained fatty acids. The substrate, MUG (4-methylumbelliferyl-β-D-glucuronide), produces a blue fluorescence when hydrolyzed by the enzyme β-glucuronidase, which is produced by most E. coli.

FORMULA

Ingredients per liter of deionized water:*

Peptone 5.0gm
Beef Extract 3.0gm
MUG (4-methylumbelliferyl-β-D-glucuronide) 0.1gm
Agar 15.0gm

Final pH 6.8 +/- 0.3 at 25°C.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store media in plates at 2-8°C. 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 and freezing.

PRECAUTIONS

PROCEDURE

Sample collection: Consult listed references for information on sample collection and procedures for water testing.(1,11)

Membrane filter-Endo LES Agar method, followed by incubation on Nutrient Agar with MUG:

1. Follow the methods and procedures for water testing using m Endo LES Agar described in Standard Methods.(1)

2. After incubation on m Endo LES Agar for 24 hours (Cat. no. G28), aseptically transfer the membrane to Nutrient Agar with MUG.(1,3,11)

3. Incubate plates at 35 +/- 0.5°C for 4 hours.

4. Expose the filter surface to long-wave (approximately 366nm) UV light, preferably containing a 6-Watt bulb.

INTERPRETATION OF RESULTS

Observe for fluorescence following incubation. Positive MUG reactions exhibit a blue fluorescence around the periphery of the colony under long-wave UV light.

Typical strains of E. coli (red with a green metallic sheen on m Endo LES Agar, Cat. no. G28) exhibit blue fluorescence on Nutrient Agar with MUG. Non-E. coli coliforms may produce a metallic sheen but do not fluoresce.

LIMITATIONS

Glucuronidase-negative strains of E. coli have been encountered.(5-7) Similarly, MUG-negative strains of E. coli have been reported in this assay procedure but at a very low frequency.(3)

Strains of Salmonella and Shigella species that produce β-glucuronidase may infrequently be encountered.(8) These strains must be distinguished from E. coli on the basis of other parameters; i.e., gas production, lactose fermentation or growth at 44.5°C.

MATERIALS REQUIRED BUT NOT PROVIDED

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

QUALITY CONTROL

The following organisms are routinely used for testing at Hardy Diagnostics:

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Escherichia coli
ATCC® 25922
A 18-24hr 35°C Aerobic Growth with fluorescence under long-wave UV light
Enterobacter aerogenes
ATCC® 13048
A 18-24hr 35°C Aerobic Growth without fluorescence under long-wave UV light

User Quality Control

PHYSICAL APPEARANCE

Nutrient Agar with MUG should appear clear to slightly opalescent, and light amber in color.

REFERENCES

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

2. Feng and Hartman. 1982. Appl. Environ. Microbiol.; 43:1320.

3. Mates and Shaffer. 1989. J. Appl. Bacteriol.; 67:343.

4. Federal Register. 1991. Fed. Regist.; 56:636.

5. Chang, Brill and Lum. 1989. Appl. Environ. Microbiol. ; 55:335.

6. Hansen and Yourassowsky. 1984. J. Clin. Microbiol. ; 20:1177.

7. Kilian and Bulow. 1976. Sect. B Acta Pathol. Microbiol. Scand. ; 84:245.

8. Damare, Campbell and Johnston. 1985. J. Food Sc.; 50:1736.

9. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

10. Pelczar, Chan and Kreig. 1986. Microbiology , 5th ed. McGraw-Hill Book Company, New York, NY.

11. Environmental Protection Agency. 2005. Manual for the Certification of Laboratories Analyzing Drinking Water, EPA-815-R-05-004. Office of Ground Water and Technical Support Division, U.S. Environmental Protection Agency, Cincinnati, OH.


ATCC is a registered trademark of the American Type Culture Collection.

051716vr