Listeria Chromogenic Agar Base according to Ottaviani and Agosti (ALOA) ISO
Principles and uses
Listeria Chromogenic Agar Base acc. to Ottaviani and Agosti (ALOA) is a selective medium for the presumptive isolation and identification of Listeria monocytogenes and Listeria spp. in food.
It is used for confirmation after using Listeria Enrichment Broth Base Fraser . This medium is also recommended by ISO 11290-1 for the detection and enumeration for Listeria monocytogenes.
Enzymatic digest of animal tissues and enzymatic digest of casein provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is the source of vitamins, particularly of the B-group. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Sodium pyruvate is a source of energy for bacterial metabolism and aids in the resuscitation of stressed organisms. Glucose is the fermentable carbohydrate providing carbon and energy. Magnesium glycerophosphate is a buffering compound. Magnesium sulphate is a magnesium ion required for a large variety of enzymatic reactions, including DNA replication. The differential activity of the medium is due to two factors. Lithium chloride in the base medium and supplementary antimicrobial compounds Ceftazidime, Polymyxin, Nalidixic acid and Cycloheximide provide the medium’s selectivity. Bacteriological agar is the solidifying agent.
The presence of the chromogenic component X-glucoside, a substrate for the detection of the enzyme ß-glucosidase, is common to all Listeria species giving the colonies their blue colour. Other organisms that possess this enzyme, for example, Enterococci, are inhibited by the selective agents within the medium and by the selective supplement. The differential activity is also obtained by lipase C substrate, upon which the specific enzyme for L. monocytogenes acts. The lipase is responsible for the opaque white halo which surrounds L. monocytogenes.
The combination of both substrates allows us to differentiate the colonies of Listeria monocytogenes from the rest of Listeria spp. since, although all are blue in colour, L. monocytogenes present an opaque white halo surrounding them. It has been observed that some strains of Listeria ivanovii, mostly pathogenic to animals although some have caused infections in humans, also possess lipase activity.
Preparation
Suspend 35,275 grams of the medium in 470 ml of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 121 ºC for 15 minutes. To prepare more quantity of 500 ml, it is recommended to sterilize at 115 ºC for 10 minutes. Cool to 47-50 ºC and aseptically add one bottle of Listeria Lipase C Supplement (24 ml) and one vial of Listeria Chromogenic Selective Supplement . Homogenize gently and dispense into Petri dishes.