MUG REAGENT

Code: BR0071

A fluorescent agent for the detection of Escherichia coli.

Directions
Add 2ml of distilled water to a vial and invert gently until completely dissolved. Add the vial contents to the following volumes of suggested media, before sterilisation.

Other media can be used, consult Oxoid for further advice.

Description
Oxoid MUG reagent is a lyophilised presentation of the substrate 4-methylumbelliferyl-ß-D-glucuronide in 50mg quantities. The incorporation of MUG reagent into culture media is reported to improve the sensitivity and specificity of E.coli detection^1,2,3,4,5. This improved sensitivity is mainly due to the detection of anaerogenic strains of E.coli when present in mixed cultures. The sensitivity reported for various media varies from 59% to 85.8%⁶. MUG reagent is cleaved by the enzyme glucuronidase to release an end product 4-methylumbelliferone which produces a visible green/blue fluorescence under long wave ultra-violet light (366 nm). The addition of MUG reagent to culture media provides another criterion by which to determine the presence of E.coli in food and environmental samples. Maddocks and Greenan⁷ adjusted the pH of their cultures with sodium hydroxide to maximise light output in their investigations of MUG hydrolysis as an alternative to conventional biochemical tests for identifying bacteria. The HPA method for the enumeration of coliforms and presumptive E.coli by the most probable number (MPN) technique suggests adding 1ml of 1M sodium hydroxide to 10ml of broth containing the MUG reagent after 48 hours incubation as a means of increasing fluorescence produced by the growth of E.coli. ¹⁴ NaOH addition generally improves fluorescence by increasing the alkalinity of the solution. An increase in fluorescence can also be achieved by increasing the level of MUG added to the base medium.

The acidification of the agar surrounding E. coli colonies on lactose-based media diminishes the discrimination of MUG-hydrolysing colonies⁸. The addition of a 10µl drop of 1M NaOH onto presumptive E.coli colonies grown on solid agar containing MUG will increase fluorescence under long wave UV light. Freir and Hartman⁹ exposed membrane filter cultures to ammonia vapour to enhance fluorescence. A phosphate-buffered MUG agar was used by Entis and Boleszczuk to minimise pH fall in an improved 24 hour hydrophobic grid membrane filter method for coliform and E. coli enumeration¹⁰.

MUG has been included in media used with membrane filters in methods for enumeration of E. coli in foods, water and sewage^11,12. Conditions that affect the fluorescence intensity of MUG were investigated by Villari, Iannuzzo and Torre and recommendations made for its optimum use¹³.

Organisms in broths or on agar, exposed to NaOH in the ways described above, may become non-viable. If further identification is likely to be required it is recommended that a sample is taken prior to addition of NaOH.

Technique

1. Follow the method and procedure relevant to the sample and the selected medium. Uninoculated tubes or agar plates should be used as controls. (See Precaution.)
2. After incubation detect glucuronidase activity by examining the microbial growth under UV light (366nm). The presence of blue/green fluorescence indicates glucuronidase activity.
3. Report fluorescence as presumptive presence of E.coli and confirm by further biochemical tests.

Storage and stability
Should be stored at 2-8°C. When stored as directed the unopened vial is stable until the expiry date on the label.

Quality control
Positive control:
Escherichia coli ATCC® 25922

Negative control:
Proteus mirabilis ATCC® 29906

Precautions
The presence of endogenous glucuronidase in shellfish samples may result in false positive fluorescence. Test tubes used in the MPN method should be checked under UV light to ensure the glass does not fluoresce. To avoid false positive fluorescence the source of long wave UV light must not exceed 6 watts.

References
1. Feng P. C. S. and Hartman P. A. (1982) Appl. Environ. Microbiol. 43. 1320-1329.
2. Harsen W. and Yourassowsky (1984) J. Clin. Microbiol. 20. 1177-1179.
3. Le Uinor L., Buissieve J., Novel G. and Novel M. (1978) Ann. Microbiol. (Paris) 129B. 155-165.
4. Kilan M. and Bulow P. (1976) Acta Pathol. Microbiol. Scand. sect B. 84. 245-251.
5. Kilan M. and Bulow P. (1979) Acta Pathol. Microbiol. Scand. sect B 87. 271-276.
6. Heizmon H. (1988) J. Clin. Microbiol. 26. 2682-2684.
7. Maddocks J. L. and Greenan M. J. (1975) J. Clin. Pathol. 28. 686-687.
8. Frampton E. W. and Restaino L. (1993) J. Appl. Bact. 74. 223-233.
9. Freir T. A. and Hartman P. A. (1987) Appl. Env. Microbiol. 53. 1246-1250.
10. Entis P. and Boleszczuk P. (1990) J. Food Prot. 53. 948-952.
11. Freir T. A. and Hartman P. A. (1987) Appl. Env. Microbiol. 53. 1246-1250.
12. Shadix L. C., Dunningan M. E. and Rice E. W. (1993) Can. J. Microbiol. 39. 1066-1070.
13. Villari P., Iannuzzo M. and Torre I. (1997) Lett. Appl. Microbiol. 24. 286-290.
14. http://www.hpa-standardmethods.org.uk/documents/milk_dairy/pdf/d5.pdf

ATCC^® is a registered trademark of American Type Culture Collection.