EC BROTH WITH MUG AND DURHAM TUBE

Cat. no. K64 EC Broth with MUG and Durham Tube, 16x125mm Tube, 10ml 20 tubes/box
Cat. no. K18 EC Broth with MUG and Durham Tube, 20x125mm Tube, 13ml 20 tubes/box

INTENDED USE

Hardy Diagnostics EC Broths with MUG and Durham Tube is recommended for the detection of Escherichia coli in water and food samples by fluorogenic means.

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

SUMMARY

EC Broth with MUG (4-methylumbelliferyl-beta-D-glucuronide) is composed of the same basal formula as EC Broth developed by Hajna and Perry. (8) The medium consists of a buffered lactose broth with casein peptones, bile salts, and MUG.

Lactose provides fermentable carbohydrate for the growth of coliforms. Casein peptones provide a source of nutrients. The bile salts serve as inhibitory agents toward gram-positive cocci and spore-formers, particularly fecal streptococci and bacilli. The pH of the medium is maintained by the presence of a strong potassium buffering system.

The addition of MUG, a fluorogenic compound, allows for the rapid detection of E. coli when the medium is observed for fluorescence using a long-wave (366nm) UV light source. (7,9) Anaerogenic strains of E. coli can also be detected through the use of MUG. (7)

The detection of E. coli with MUG is based on the ability of beta-glucuronidase, an enzyme possessed by most E. coli strains, to hydrolyze 4-methylumbelliferyl-beta-D-glucuronide. The hydrolysis of MUG by E. coli yields 4-methylumbelliferone, a fluorescent end product. (7,9) Development of fluorescence allows the detection of E. coli in pure or mixed cultures within 4 to 24 hours following inoculation and incubation of EC Broth with MUG.

Studies conducted by Feng and Hartman revealed beta-glucuronidase activity in 96% of E. coli , 100% of enterotoxigenic E. coli , 17% Salmonella spp. and 40% of Shigella spp. (7)

EC Broth is recommended by the American Public Health Association (APHA) for the detection and enumeration of coliform organisms in foods, waters, and wastewater. (1,2)

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 20.0gm
Lactose 5.0gm
Sodium Chloride 5.0gm
Dipotassium Phosphate 4.0gm
Monopotassium Phosphate 1.5gm
Bile Salts Mixture 1.5gm
MUG 50.0mg

Final pH 6.9 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt, store the product at 2-8ºC. Products should not be used if there are any signs of contamination, deterioration, 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. (1-5)

Method of Use: Allow medium to warm to room temperature prior to inoculation. Consult listed references for information concerning inoculation procedures. (1-5)

Incubate tubes in a 44.5 +/- 0.2ºC. waterbath for water and wastewater. Incubate tubes in a waterbath at 45.5 +/- 0.2ºC. for foods other than shellfish.

INTERPRETATION OF RESULTS

Production of turbidity and gas within 24 +/- 2 hours of incubation without fluorescence is considered positive evidence of fecal coliforms in water and wastewater. Presence of growth and the production of a bright blue fluorescence under a long-wave UV light (with or without the production of gas) is considered positive for the presence of E. coli . Absence of gas production within 24 +/- 2 hours is considered a negative test for fecal coliforms.

Production of turbidity and gas within 48 +/- 2 hours of incubation without fluorescence is considered considered positive evidence of fecal coliforms in foods other than shellfish. Presence of growth and the production of a bright blue fluorescence (with or without gas production) under a long-wave UV light is considered positive for the presence of E. coli . Absence of gas production within 48 +/- 2 hours is considered a negative test for fecal coliforms.

Consult listed references for detailed results for the enumeration of coliforms using EC Broth with MUG.

LIMITATIONS

It may be necessary to invert the tube prior to inoculation if bubbles are trapped in the durham tube. Trapped bubbles that are not released may lead to false-positive results.

Turbidity alone is not indicative of a positive test for the presence of fecal coliforms; turbidity with gas production is considered a positive test for fecal coliforms. Fluorescence may be observed in anaerogenic E. coli .

An uninoculated tube of media should be included as a batch control to detect weak autofluorescence of the medium.

Some strains of Shigella and Salmonella produce beta-glucuronidase which may result in false interpretation of test results.

The presence of streptococci in the test sample may lead to false-positive results.

Enterobacter aerogenes will not produce gas, and growth may be reduced, when incubated at 44.5°C.

False-positive results may occur when testing oysters. Oysters produce glucuronidase which interferes with the accuracy of the assay. When testing oyster samples, it is recommended that an enrichment step in Lauryl Sulfate Broth be performed prior to inoculation of the test sample to EC Broth with MUG. The preenrichment step dilutes the glucuronidase from the oysters and decreases the possibility of false-positive results.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, Lauryl Sulfate Broth, other culture media, swabs, applicator sticks, 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**
A 24hr 35°C Aerobic Growth; turbidity with gas production and fluorescence under a long-wave UV light
Enterobacter aerogenes
ATCC® 13048**
A 24hr 35°C Aerobic Growth; turbidity with gas and no fluorescence
Enterococcus faecalis
ATCC® 29212**
B 24hr 35°C Aerobic Inhibited

**Recommended QC strains.

User Quality Control

PHYSICAL APPEARANCE

Both EC Broths with MUG and Durham Tube should appear clear, and colorless to light amber in color.

E. coli (LEFT) and E. aerogenes (RIGHT) growing in EC Broth with MUG under UV light

Escherichia coli (ATCC® 25922) (LEFT) and Enterobacter aerogenes (ATCC® 13048) (RIGHT) growing in EC Broth with MUG (Cat. no. K18) under UV light. Incubated aerobically for 24 hours at 35ºC.

E. coli growing in EC Broth with MUG

Escherichia coli (ATCC® 25922) growing in in EC Broth with MUG (Cat. no. K18) under ambient light. The bubble in the Durham tube indicates gas production. Incubated aerobically for 24 hours at 35ºC.

E. faecalis inhibited in EC Broth with MUG under UV light

Enterococcus faecalis (ATCC® 29212) growth inhibited in EC Broth with MUG (Cat. no. K18) under ambient light. Incubated aerobically for 24 hours at 35ºC.

REFERENCES

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

2. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods. APHA, Washington, D.C.

3. American Public Health Association. Standard Methods for the Examination of Dairy Products. APHA, Washington, D.C.

4. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA. www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm

5. Association of Official Analytical Chemists. Official Methods of Analysis. AOAC, Washington, D.C.

6. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology. J.B. Lippincott Company, Philadelphia, PA.

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

8. Hajna and Perry. 1943. Am. J. Public Health; 33:550.

9. Robison. 1984. Appl. Environ. Microbiol.; 48:285.


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

021816sw