Cronobacter Selective Broth (CSB) ISO

Enzymatic digest of animal tissues and meat extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Sucrose is the fermentable carbohydrate providing carbon and energy. Bromocresol purple is a pH indicator.

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.

ISO 22964 FDIS:2016 describes a horizontal method for the detection of Cronobacter spp. and recommends this medium for the enrichment of Cronobacter spp. in food, in animal feed and in environmental samples. A yellow/orange coloration in the broth after incubation (due to sucrose fermentation) indicates presumptive Cronobacter. If the broth is still purple in colour after incubation, the sample is presumed to be negative for Cronobacter.

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

Preparation

Suspend 28.04 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. Sterilize in autoclave at 121 °C for 15 minutes. Cool to 50 °C and aseptically add 2 vials of Vancomycin Supplement (LC-6072) so as to obtain a final vancomycin concentration of 10 mg per litre of CSB. Homogenize gently and dispense into tubes in amounts of 10 mL.

According to ISO 22964

For the detection of Cronobacter spp. according to ISO 22964:

1. Prepare the test portion by adding 10 g or 10 mL of the sample to 90 mL of BPW pre-enrichment medium.
2. Incubate at 34-38 °C for 18 ± 2 hours.
3. Transfer 0.1 mL of the obtained culture to 10 mL of CSB and mix well.
4. Incubate at 41.5 ± 1 °C for 24 ± 2 hours.
5. Using the previous enrichment culture, inoculate the surface of a CCI Agar (LC-1446) and incubate at 41.5 ± 1 °C for 24 ± 2 hours.
6. Confirmation.

• ISO 22964:2016 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.