Part of Thermo Fisher Scientific
Organisms in the Industrial sector this product works with:
Other products used in the isolation of Escherichia coli:
MUG REAGENT
Code: BR0071
A fluorescent agent for the detection of Escherichia coli.
Vial content | |
4-methylumbelliferyl-ß-D-glucuronide | 50mg |
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.
Medium | Final conc. of MUG per litre | Number of vials per litre |
Violet Red Bile Agar CM0107 | 100mg | 2 |
MacConkey Agar No. 3 CM0115 | 100mg | 2 |
Brilliant Green Bile (2%) Broth CM0031 | 50mg | 1 |
MacConkey Broth Purple CM0505 | 50mg | 1 |
Lauryl Tryptose Broth CM0451 | 50mg | 1 |
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 detection1,2,3,4,5. 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 sewage11,12. 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
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.