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Technical Data
EMB Agar, Levine M022
Intended Use:
Recommended for the isolation, enumeration and differentiation of members of Enterobacteriaceae from clinical and non
clinical samples.
Composition**
Ingredients g/L
Peptone 10.000
Dipotassium hydrogen phosphate 2.000
Lactose 10.000
Eosin - Y 0.400
Methylene blue 0.065
Agar 15.000
Final pH ( 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.
Precaution : Store the medium away from light to avoid photo-oxidation.
Principle And Interpretation
Levine EMB Agar was developed by Levine (1,2) and is used for the differentiation of Escherichia coli and Klebsiella
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 (3,4,5). Weld
(6,7) 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.
Eosin Y and methylene blue make the medium slightly selective and inhibit certain gram-positive bacteria. These dyes serve
as differential indicators in response to the fermentation of carbohydrates. This helps to differentiate between lactose-
fermenters and non-fermenters in EMB Agar, Levine. The ratio of eosin-methylene blue is adjusted to approximately 6:1.
Coliforms produce purplish black colonies due to uptake of methylene blue-eosin dye complex, when the pH drops. The
dye complex is absorbed into the colony. Non-fermenters probably raise the pH of surrounding medium by oxidative
de-amination of protein, which solubilizes the methylene blue-eosin complex resulting in formation of colourless
colonies . Peptone serves as source of carbon, nitrogen, long chain amino acids, vitamins and other essential growth
nutrients. Lactose serves as the source of energy by being the fermentable carbohydrate. Eosin-Y and methylene
blue serve as differential indicators. Phosphate buffers the medium.
The test sample can be directly streaked on the medium plates. Inoculated plates should be incubated, protected from light.
However standard procedures should be followed to obtain isolated colonies. A non-selective medium should be inoculated
in conjunction with EMB Agar. Confirmatory tests should be further carried out for identification of isolated colonies.
Type of specimen
Clinical samples - urine, faeces, oral and vaginal secretions and nail or skin scraping , Foodstuffs; Water samples.
Specimen Collection and Handling
For clinical samples follow appropriate techniques for handling specimens as per established guidelines (8,9).
For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (5,10).
For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (3).
After use, contaminated materials must be sterilized by autoclaving before discarding.
Please refer disclaimer Overleaf.
HiMedia Laboratories Technical Data

Warning and Precautions

In Vitro diagnostic use. For professional use only. Read the label before opening the container. Wear protective
gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling
specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical
specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations
1. A non-selective medium should be inoculated in conjunction with EMB Agar.
2. Confirmatory tests should be further carried out for identification of isolated colonies.
3. Some strains of Salmonella and Shigella species do not grow in the presence of eosin and methylene blue.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when
stored at recommended temperature.
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 coloured, opalescent gel with greenish cast and finely dispersed precipitate forms in Petri plates
Reaction
Reaction of 3.75% w/v aqueous solution at 25°C. pH : 7.1±0.2
pH
6.90-7.30
Cultural Response
Cultural characteristics observed after an incubation at 35-37°C for 24-48 hours.
Organism Inoculum Growth Recovery Colour of
(CFU) Colony
Candida albicans ATCC 50-100 luxuriant >=50% colourless
10231 (00054*) (incubated in
10% carbon
dioxide)
# Klebsiella aerogenes 50-100 good 40-50% pink-red
ATCC 13048 (00175*)
Escherichia coli ATCC 50-100 luxuriant >=50% blue-black with
25922 (00013*) metallic sheen
Enterococcus faecalis 50-100 none-poor <=10% colourless
ATCC 29212 (00087*)
Pseudomonas aeruginosa 50-100 luxuriant >=50% colourless
ATCC 27853 (00025*)
Staphylococcus aureus 50-100 none-poor <=10% colourless
subsp. aureus ATCC
6538 (00032*)
^Pseudomonas paraeruginosa 50-100 luxuriant >=50% colourless
ATCC 9027 (00026*)
50-100
Salmonella Typhimurium luxuriant >=50% colourless
ATCC 14028 (00031*)
Saccharomyces cerevisiae 50-100 none-poor <=10% cream
ATCC 9763
Staphylococcus aureus 50-100 none-poor <=10% colourless
subsp. aureus ATCC 25923
(00058*)
blue-black with
Escherichia coli ATCC 50-100 luxuriant >=50% green metallic
8739 (00012*)
sheen

Key : (*) Corresponding WDCM numbers.

(#) Formerly known as Enterobacter aerogenes ^ Formerly known as Pseudomonas aeruginosa

Please refer disclaimer Overleaf.

HiMedia Laboratories Technical Data

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on
the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump
formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store
in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use.
Product performance is best if used within stated expiry period.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow
established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical
sample must be decontaminated and disposed of in accordance with current laboratory techniques (8,9).
Reference
1. Levine M., 1918, J. Infect. Dis., 23:43.
2. Levine M., 1921, Bull. 62, Iowa State College Engr. Exp. Station.
3. Lipps WC, Braun-Howland EB, Baxter TE,eds. Standard methods for the Examination of Water and Wastewater, 24th ed.
Washington DC:APHA Press; 2023.
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., 2015, Compendium of Methods for the Microbiological Examination of Foods, 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. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
9. 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.
10. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed.,
Washington D.C.

Revision : 06/2024

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User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in this and
other related HiMedia™ publications. The information contained in this publication is based on our research and development work and is to the best
of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to specifications and information related
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