Plot No. C40, Road No. 21Y, MIDC, Wagle Industrial Estate,
Thane(W)-400604, MH, India.
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EMB Agar, Levine (Levine - Eosin Methylene Blue Agar Medium) (In accordance with IP 2007)
For isolation, enumeration and differentiation of members of Enterobacteriaceae in accordance with Indian Pharmacopoeia.
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Pharmaceutical Laboratory Testing#CC293D
Intended Use
Levin Eosin Methylene Blue Agar Medium is recommended for the isolation, enumeration and differentiation of members of Enterobacteriaceae in accordance with Indian Pharmacopoeia..
Composition
| Ingredients | Gms/Litre |
|---|---|
| Pancreatic digest of gelatin | 10.000 |
| Dibasic potassium phosphate | 2.000 |
| Lactose | 10.000 |
| Eosin - Y | 0.400 |
| Methylene blue | 0.065 |
| Agar | 15.000 |
| pH after sterilization (at 25°C) | 7.1±0.2 |
Formula adjusted, standardized to suit performance parameters
Directions
Suspend 37.46 grams in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes or as per validated cycle. AVOID OVERHEATING. Cool to 50°C and shake the medium in order to oxidize the methylene blue (i.e. restore its blue colour) and to suspend the precipitate, which is an essential part of the medium. Mix well before pouring into sterile Petri plates.
Precaution: Store the medium away from light to avoid photooxidation.
Principle And Interpretation
Levin Eosin Methylene Blue Agar was developed by Levine (1,2) and is used for the differentiation of Escherichia coli and Enterobacter aerogenes and also for the rapid identification of Candida albicans. This medium is recommended for the detection, enumeration and differentiation of members of the coliform group by American Public Health Association and Indian Pharmacopoeia (3, 4, 5, 6).
Eosin-Y and methylene blue make the medium slightly selective and inhibit certain gram-positive bacteria. Both dyes act as indicator and inhibiting agent. These dyes differentiate between lactose fermenters and non-fermenters. Eosin Y and methylene blue forms a complex at acidic pH which acts as inhibiting agent. Some gram-positive bacteria such as faecal Streptococci, yeasts grow on this medium and form pinpoint colonies. Essential nutrients and growth factors is provided by pancreatic digest of gelatin. Phosphates act as good buffering agent. E.coli forms colonies with green metallic sheen, indicating strong lactose fermentation. Weld (7,8) proposed the use of Levine EMB Agar, with added Chlortetracycline hydrochloride, for the rapid identification of Candida albicans in clinical specimens. A positive identification of Candida albicans can be made after 24 48 hours incubation at 35 - 37°C in 10% carbon dioxide atmosphere, from specimens such as faeces, oral and vaginal secretions and nail or skin scraping etc. However, the typical appearance is variable.
Quality Control
Appearance: Light pink to purple homogeneous free flowing powder
Gelling: Firm, comparable with 1.5% Agar gel
Colour and Clarity of prepared medium: Reddish purple with greenish cast coloured opalescent gel with finely dispersed precipitate forms in Petri plates.
pH: 6.90-7.30
Cultural Response
Growth Promotion is carried out in accordance with IP. Cultural response was observed after an incubation at 36-38°C for 18-24 hours. Recovery rate is considered as 100% for bacteria growth on Soyabean Casein Digest Agar.
| Organism | Inoculum (CFU) | Observed Lot Recovery value (CFU) | Colour of Colony | Incubation temperature | Incubation period |
|---|---|---|---|---|---|
| Test for specified microorganism | |||||
| Escherichia coli ATCC 8739 | 50-100 | 25-100 | >=50% | blue-black colonies with metallic sheen | 36-38 °C |
| Escherichia coli NCTC 9002 | 50-100 | 25-100 | >=50% | blue-black colonies with metallic sheen | 36-38 °C |
| Additional Microbiological testing | |||||
| Escherichia coli ATCC 25922 | 50-100 | 25-100 | >=50% | blue-black colonies with metallic sheen | 36-38 °C |
| Enterobacter aerogenes ATCC 13048 | 50-100 | 25-100 | >=50% | pink to red | 36-38 °C |
| Salmonella Typhimurium ATCC 14028 | 50-100 | 25-100 | >=50% | colourless | 36-38 °C |
| Pseudomonas aeruginosa ATCC 9027 | 50-100 | 25-100 | >=50% | colourless | 36-38 °C |
| Enterococcus faecalis ATCC 29212 | >=10³ | 0 | 0% | 36-38 °C | |
| Staphylococcus aureus ATCC 6538 | >=10³ | 0 | 0% | 36-38 °C | |
| Candida albicans ATCC 10231 | 50-100 | 25-100 | >=50% | colourless | 36-38 °C |
| Saccharomyces cerevisiae ATCC 9763 | 50-100 | 0-10 | 0-10% | cream | 36-38 °C |
Storage and Shelf Life
Store below 30°C in tightly closed container and prepared medium at 2 - 8°C and away from light. Use before expiry date on the label.
| Product Name | EMB Agar, Levine (Levine - Eosin Methylene Blue Agar Medium) (In accordance with IP 2007) |
|---|---|
| SKU | MM022 |
| Product Type | Regular |
| Physical Form | Powder |
| Origin | Animal |
| Packaging type | HDPE |
| References | 1.Levine M., 1918, J. Infect. Dis., 23:43. 2.Levine M., 1921, Bull. 62, Iowa State College Engr. Exp. Station. 3.Eaton A. D., Clesceri L. S. and Greenberg A W.,(Eds.), 2005, Standard Methods for the Examination of Water andWastewater, 21st ed., APHA, Washington, D.C. 4.Wehr H M and Frank J H., 2004, Standard Methods for the Examination of Dairy Products, 17th ed., APHA Inc., Washington,D.C. 5.Downes F P and Ito K. (Eds.), 2001, Compendium of Methods for the Microbiological Examination of Foods, 4th ed., APHA,Washington, D.C. 6.Indian Pharmacopoeia, 2007, Govt. of India, Ministry of Health and Family Welfare, New Delhi. 7.Weld J. T., 1952, Arch. Dermat. Syph., 66:691. 8.Weld J. T., 1953, Arch. Dermat. Syph., 67(5):433. |
| Customized Product Available | No |
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