EC Medium with MUG

Intended Use

EC Medium with MUG is used for detecting Escherichia coli in water, food and milk.

Summary and Explanation

EC Medium was developed by Hajna and Perry¹ to improve the methods for the detection of coliforms and E. coli. This medium consists of a buffered lactose broth with the addition of 0.15% Bile Salts No. 3. Growth of sporeformers and fecal streptococci is inhibited by the bile salts, while growth of E. coli is enhanced. EC Medium with MUG is the same formula as EC Medium with the addition of 4 methyl-umbelliferyl- β-D-glucuronide.
Feng and Hartman² developed a rapid assay for E. coli by incorporating 4-methylumbelliferyl-β-D-glucuronide (MUG) into Lauryl Tryptose Broth at a final concentration of 100 μg/mL. Robison³ compared the fluorogenic assay with present methodology and found that total agreement between the two methods was 94.8%. Moburg⁴ determined the amount of MUG could be reduced to a final concentration of 50 μg/ mL without adversely affecting results. Koburger and Miller⁵ recommended the incorporation of MUG into EC Broth for use in testing shellfish.
EC Medium with MUG is prepared according to the formula specified by the U.S. Environmental Protection Agency⁶ and standard methods for water and food testing.^7,8

Principles of the Procedure

Peptone provides the nitrogen, vitamins and amino acids in EC Medium with MUG. Lactose is the carbon source in this medium. Bile Salts No. 3 is the selective agent against grampositive bacteria, particularly bacilli and fecal streptococci. Dipotassium phosphate and monopotassium phosphate are buffering agents. Sodium chloride maintains the osmotic balance of the medium.
E. coli produces the enzyme glucuronidase that hydrolyzes MUG to yield a fluorogenic product that is detectable under long wave (366 nm) UV light. The addition of MUG to EC Medium provides another criterion, in addition to growth response and gas production, to determine the presence of E. coli in food and environmental samples.

User Quality Control

Identity Specifications
EC Medium with MUG
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution:                        3.71% solution, soluble in purified water. Solution is light amber, clear.
Prepared Appearance:    Light amber, clear.
Reaction of 3.71%
Solution at 25°C:            pH 6.9 ± 0.2
Cultural Response
EC Medium with MUG
Prepare the medium per label directions. Inoculate tubes in duplicate with fresh 18-24 hour cultures. Incubate the first set at 35 ± 2°C for 24 ± 2 hours and the second set at 44.5 ± 0.2°C for 24 ± 2 hours. Read fluorescence under a long-wave UV light.

Formula

EC Medium with MUG
Approximate Formula* Per Liter
Tryptose..................................................................... 20.0 g
Lactose........................................................................ 5.0 g
Bile Salts No. 3............................................................. 1.5 g
Dipotassium Phosphate.................................................. 4.0 g
Monopotassium Phosphate............................................. 1.5 g
Sodium Chloride........................................................... 5.0 g
MUG (4-methylumbelliferyl-β-D-glucuronide)................. 0.05 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Dissolve 37.1 g of the powder in 1 L of purified water. Mix thoroughly.
2. Warm slightly to completely dissolve the powder.
3. Dispense into test tubes containing inverted fermentation vials.
4. Autoclave at 121°C for 15 minutes.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Follow the methods and procedures as stated in appropriate references.^6-8

Expected Results

Following incubation, observe tubes for growth, production of gas and fluorescence. Positive gas production is demonstrated by displacement of the medium from the fermentation vial. Positive MUG reactions exhibit a bluish fluorescence under long-wave (approximately 366 nm) UV light. Typical strains of E. coli are positive for both gas production and fluorescence. Non-E. coli coliforms that grow may exhibit fluorescence but will not produce gas.
Strains of Salmonella, Shigella and Yersinia that produce glucuronidase may be encountered. These strains must be distinguished from E. coli on the basis of other parameters; i.e., gas production, growth at 44.5°C.

Limitations of the Procedure

1. Strains of E. coli that fail to grow in EC Medium with MUG, fail to produce gas, or fail to produce glucuronidase may infrequently be encountered.
2. The presence of endogenous glucuronidase in shellfish samples may cause false positive fluorescent reactions at the presumptive stage. To prevent this problem, the use of EC Medium with MUG in the confirmatory stage has been recommended.⁵

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