L-Ornithine Decarboxylation Medium ISO

ISO 22964 describes a horizontal method for the detection of Cronobacter spp. and recommends this medium for the biochemical confirmation of Cronobacter spp.

Cronobacter (formerly Enterobacter sakazakii) is currently considered an emerging pathogen responsible for severe meningitis and necrotic enterocolitis in un-weaned babies that can be the cause of a mortality rate between 40-80%. The pathogenicity of Cronobacter for un-weaned babies makes it necessary to review the manufacturing process of the milk-based products specialized for babies, guaranteeing the absence of the bacteria in the final product.

Additional prevention measures at a hospital include the sanitary hygiene of the prepared food and reducing the time between the preparation and its administration, to impede the multiplication of microorganisms.

Yeast extract provides nitrogen, minerals, amino acids and vitamins essential for growth, particularly of the B-group. Glucose is the fermentable carbohydrate providing carbon and energy. L-ornithine is added to test the presence of the enzyme ornithine decarboxylase. If the organisms possess such enzyme, it will be activated in an acid environment created by the initial fermentation of glucose. Once the amino acid is decarboxylated, the diamine putrescine is produced. The result is an alkalinization of the medium, which turns it purple or violet. Organisms without the enzyme will remain acidic due to the fermentation, resulting in a yellow color in the medium. Bromocresol purple is a pH indicator to indicate decarboxylase activity.

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 14.0 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into tubes and sterilize in autoclave at 121 °C for 15 minutes.

Instructions for use

1. Pre-enrich the test portion in a non-selective medium, Buffered Peptone Water BPW (LC-1402).
2. Inoculate the culture obtained in BPW in the selective enrichment medium Cronobacter Selective Broth (CSB) (LC-2143).
3. Plate out and identify the colonies in the Chromogenic Cronobacter Isolation Agar (CCI) (LC-1446).
4. For confirmation, typical colonies are selected from the chromogenic agar, purified on a non-selective agar such as TSA (LC-1068) and biochemically characterized.
5. Inoculate the L-Ornithine Decarboxylation Medium with each of the selected colonies to observe the decarboxylation of the L-ornithine.

• ISO 22964 Microbiology of the food chain - Horizontal method for the detection of Cronobacter spp.
• GUILLAUME-Gentil, O., Sonnard, V. Kandahai, M.C., Mauragg, J.D. and Jootsen, H. A simple and Rapid Cultural Method for Detection of Enterobacter sakazakii in environmental samples. Journal of Food Protection, 68 (1), 2005, pp. 64-69.