EOSIN METHYLENE BLUE AGAR (MODIFIED) LEVINE

Code: CM0069

An isolation medium for the differentiation of the Enterobacteriaceae.

* Adjusted as required to meet performance standards

Directions
Suspend 37.5g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 60°C and shake the medium in order to oxidise the methylene blue (i.e. restore its blue colour) and to suspend the precipitate which is an essential part of the medium.

Description
This versatile medium, modified by Levine^1,2, is used for the differentiation of Escherichia coli and Enterobacteria aerogenes, for the rapid identification of Candida albicans, and for the identification of coagulase-positive staphylococci.

The medium is prepared to the formula specified by the APHA^3,4,5,6 for the detection and differentiation of the coliform group of organisms^7,8.
Weld ^9,10 proposed the use of Levine eosin methylene blue agar, with added chlortetracycline hydrochloride for the rapid identification of Candida albicans in clinical materials. A positive identification of Candida albicans could be made after 24 to 48 hours incubation at 37°C in 10% carbon dioxide from faeces, oral and vaginal secretions, and nail or skin scrapings. Vogel and Moses ¹¹ confirmed the reliability of Weld’s method for the relatively rapid identification of Candida albicans in sputum. They found that use of eosin methylene blue agar was just as reliable as more conventional methods for the identification of this organism in sputum. In addition, the medium provided a means for the identification of several Gram- negative genera. Doupagne¹² also investigated the use of the Levine medium under tropical conditions.

Haley and Stonerod ¹³ found that Weld’s method was variable so that Walker and Huppert ¹⁴ advocated the use of corn meal agar and a rapid fermentation test in addition to the Levine medium. Using the combined rapid technique they were able to obtain results within 48 to 72 hours.
Subsequent to the findings of Vogel and Moses ¹¹, Menolasino et al.¹⁵ used Levine eosin methylene blue agar for the identification of coagulase-positive staphylococci which grew as characteristic colourless, pin-point colonies. The Levine medium was more efficient than tellurite glycine agar and showed good correlation with the plasma coagulase test.

Colonial Characteristics
Escherichia coli- isolated colonies, 2-3mm diameter, with little tendency to confluent growth, exhibiting a greenish metallic sheen by reflected light and dark purple centres by transmitted light.
Enterobacter aerogenes - 4-6mm diameter, raised and mucoid colonies, tending to become confluent, metallic sheen usually absent, grey-brown centres by transmitted light.
Non-lactose fermenting intestinal pathogens - translucent and colourless
Candida albicans - after 24 to 48 hours at 35°C in 10% carbon dioxide `spidery’ or `feathery’ colonies. Other Candida species produce smooth yeast-like colonies. Since a typical appearance is variable it is advisable to use a combined method such as that of Walker and Huppert ¹⁴.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates at 2-8°C away from light.

Appearance
Dehydrated medium: purple coloured, free-flowing powder
Prepared medium: dark purple gel

Quality control

* This organism is available as a Culti-Loop®

Precautions
Further tests are required to confirm the presumptive identity of organisms isolated on this medium. Some strains of Salmonella and Shigella species will not grow in the presence of eosin and methylene blue. Store the medium away from light to prevent photo-oxidation.

References
1. Levine M. (1918) J. Infect. Dis. 23. 43-47.
2. Levine M. (1921) `Bacteria Fermenting Lactose and the Significance in Water Analysis’ Bull. 62. Iowa State College Engr. Exp. Station.
3. American Public Health Association (1980) Standard Methods for the Examination of Water and Wastewater. 15th Edn. APHA Inc. Washington DC.
4. American Public Health Association (1978) Standard Methods for the Examination of Dairy Products. 14th Edn. APHA Inc. Washington DC.
5. American Public Health Association (1992) Compendium of Methods for the Microbiological Examination of Foods 3rd Edn. APHA Inc. Washington DC.
6. American Public Health Association (1970) `Diagnostic Procedures’. 5th Edn. APHA Inc. Washington DC.
7. American Society for Microbiology (1974) Manual of Clinical Microbiology 2nd Edn. ASM Washington DC.
8. Windle Taylor E. (1958) `The Examination of Waters and Water Supplies’ 7th Ed., Churchill Ltd., London.
9. Weld Julia T. (1952) Arch. Dermat. Syph. 66. 691-694.
10. Weld Julia T. (1953) Arch. Dermat. Syph. 67(5). 473-478.
11. Vogel R. A. and Moses Mary R. (1957) Am. J. Clin. Path. 28. 103-106.
12. Doupagne P. (1960) Ann. Soc. Belge de Med. Trop. 40(6). 893-897.
13. Haley L. D. and Stonerod M. H. (1955) Am. J. Med. Tech. 21. 304-308.
14. Walker Leila and Huppert M. (1959) Am. J. Clin. Path. 31. 551-558.
15. Menolasino N. J., Grieves Barbara, Payne Pearl (1960) J. Lab. Clin. Med. 56. 908-910.