TITAN MEDIA
EMB AGAR, LEVINE (VEG.)
TMV 371
EMB AGAR, LEVINE (VEG.) For isolation, enumeration and differentiation of Enterobacteriaceae
Composition Ingredients
Gms/Ltr.
Agar
15.00
Veg. Peptone Lactose Dipotassium phosphate Eosin Y Methylene blue
10.00 10.00 2.00 0.40 0.075
* Dehydrated powder, hygroscopic in nature, store, in a dry place in tightly- sealed containers below 25°C and protect from direct Sunlight.
Instructions for Use Dissolve 37.50gms of the medium in 1000ml of purified water or distilled water. Gently heat to boiling with gentle swirling and dissolve the medium completely. Sterilize by autoclaving at 15psi (at 121°C) for 15 minutes. DO NOT OVER HEAT. Cool to 45-50°C and swirl gently to pour into sterile Petri plates. Appearance: Dark red to blue purple colour, with or without fine precipitate 0
pH (at 25 C): 7.1 ± 0.2
Principle EMB AGAR or EOSIN METHYLENE BLUE AGAR, LEVINE (VEG.) is used for isolation, enumeration and differentiation of Enterobacteriaceae. First EMB was introduced by Holt - Harris and Teague in 1916 to differentiate Escherichia spp. and Aerobacterspp. Later, modified by Levine in 1918 who has increased the Lactose content by removing Sucrose from the formula. The medium contains Veg. Peptone source of carbon and nitrogen for the growth of microorganisms. Lactose is the fermentable carbohydrates. Differentiation of enteric bacteria is possible due to the presence of the sugars lactose in the EMB agar and the ability of certain bacteria to ferment lactose in the medium. Lactose-fermenting gram-negative bacteria (generally enteric) acidify the medium, and under acidic conditions the dyes produce a dark purple complex which is usually associated with a green metallic sheen. Dipotassium phosphate is the buffer. Eosin Y and Methylene blue are dyes that combine to form a complex at an acid pH. Small amounts of this dye effectively inhibit the growth of most gram-positive bacteria. Eosin is a dye that responds to changes in pH, going from colorless to black under acidic conditions. At a sufficiently low pH, strong lactose fermenters such as Escherichia coli produce colonies with a green metallic sheen. This metallic green sheen is an indicator of vigorous lactose fermentation ability typical of fecal coliforms. A smaller amount of acid production, which is a result of slow fermentation (by slow lactose-fermenting organisms), gives a brown-pink coloration of growth. Colonies of non-lactose fermenters appear as translucent or pink. Agar is the solidifying agent.
Interpretation Cultural characteristics observed after inoculation (10 Microorganism
3-
5
10 CFU/ml), on incubation at 35°C for 24 - 48 hours.
Inoculum
Recovery
(CFU/ml)
rate (%)
ATCC
Growth
Appearance of colony
TITAN MEDIA
25922
10
3
>=50%
Escherichia coli Salmonella typhimurium
Blue - black with green Luxuriant
3
14028
10
29212
10 10
27853
10
3-
5
>=50%
Luxuriant
metallic sheen Colourless
<=10%
Partially
Colourless, pin point
>=50%
inhibited Luxuriant
colonies Colourless
Enterococcus faecalis Pseudomonas aeruginosa
3
References 1. Holt-Harris, J. E., and O. Teague. A new culture medium for the isolation of Bacillus typhosa from stools. J. Infect. Dis. 18: 596. (1916). 2. Levin, M. Differentiation of E. coli and A. aerogenes on a simplified eosin-methylene blue agar. J. Infect. Dis. 23:43-47. (1918). 3. Levin, M. Bacteria fermenting lactose, the significance in water analysis. Bull. 62. Iowa State College Eng. Exp. Sta., Ames, Iowa. (1921). th
4. Cunnif, P. (ed.). Official Methods of Analysis AOAC International, 16 ed. AOAC International, Gaithersburg, MD. (1995). th
5. U.S. Food and Drug Administration. Bacteriological analytical manual, 8 ed. AOAC International, Gaithersburg, MD. (1995). 6. Vanderzant, C., and D. F. Splittstoesser (eds.). Compendium of methods for the microbiological rd examination of food., 3 ed. American Public Health Association, Washington, D.C. (1992). th
7. Marshall, R. T. (ed.). Standard methods for the microbiological examination of dairy products, 16 ed. American Public Health Association, Washington, D.C. (1993). 8. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (eds.). Manual of clinical th microbiology, 6 ed. American Society for Microbiology, Washington, D.C. (1995).
9. MacFaddin, J.F. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD. (1985).
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