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Levine Eosin - Methylene Blue Agar Medium

Name: Levine Eosin - Methylene Blue Agar Medium
Brand: HiMedia Laboratories
SKU: MU022
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MU022

For isolation, enumeration and differentiation of members of Enterobacteriaceae in accordance with US Pharmacopoeia.

Description

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. 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

Levine Eosin Methylene Blue Agar Medium 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 United States Pharmacopoeia (3, 4, 5, 6).

Eosin-Y and methylene blue makes 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. Essential nutrients and growth factors are provided by pancreatic digest of gelatin. Phosphates acts 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. Some gram-positive bacteria such as faecal Streptococci, yeasts grow on this medium and form pinpoint colonies 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 USP. Cultural response was observed after an incubation at 30-35°C for 24-48 hours. Recovery rate is considered as 100% for bacteria growth on Soyabean Casein Digest Agar and fungal growth on Sabouraud Dextrose 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	30-35 °C	24-48 hrs
Additional Microbiological testing
Escherichia coli NCTC 9002	50-100	25-100	>=50%	blue-black colonies with metallic sheen	30-35 °C	24-48 hrs
Escherichia coli ATCC 25922	50-100	25-100	>=50%	blue-black colonies with metallic sheen	30-35 °C	24-48 hrs
Enterobacter aerogenes ATCC 13048	50-100	25-100	>=50%	pink to red	30-35 °C	24-48 hrs
Salmonella Typhimurium ATCC 14028	50-100	25-100	>=50%	colourless	30-35 °C	24-48 hrs
Pseudomonas aeruginosa ATCC 9027	50-100	25-100	>=50%	colourless	30-35 °C	24-48 hrs
Enterococcus faecalis ATCC 29212	>=10³	0	0%	-	30-35 °C	24-48 hrs
Staphylococcus aureus ATCC 6538	>=10³	0	0%	-	30-35 °C	24-48 hrs
Candida albicans ATCC 10231	50-100	25-100	>=50%	colourless	30-35 °C	24-48 hrs
Saccharomyces cerevisiae ATCC 9763	50-100	0-10	0-10%	cream	30-35 °C	24-48 hrs

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 Information

More Information
Product Name	Levine Eosin - Methylene Blue Agar Medium
SKU	MU022
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.Baird R.B., Eaton A.D., and Rice E.W., (Eds.), 2015, Standard Methods for the Examination of Water and Wastewater,23rd ed., APHA, Washington, D.C. 4.Marshall R. (Ed.), 1992, Standard Methods for the Examination of Dairy ,, Products, 16th ed., APHA Inc., New York. 5.Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination ofFoods, 5th Ed., American Public Health Association, Washington, D.C. 6.Weld J. T., 1952, Arch. Dermat. Syph., 66:691. 7.Weld J. T., 1953, Arch. Dermat. Syph., 67(5):433. 8.Howard B. J., 1994, Clinical and Pathogenic Microbiology, 2nd Ed., Mosby Year Book, Inc9.American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed.,Washington D.C. 10.Downes F. P. and Ito K., (Ed.), 2001, Compendium of Methods for the Microbiological Examination of Foods, 4th Ed.,American Public Health Association, Washington, D.C. 11. Isenberg, H.D. Clinical Microbiology Procedures Handb0ook. 2nd Edition.12 Jorgensen,J.H., Pfaller , M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
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