Chromogenic medium for detection of Cronobacter spp, according to ISO 22964

DESCRIPTION
ChromaticTM Cronobacter Isolation Agar (CCI) is a chromogenic medium used for the isolation of Cronobacter spp (formerly Enterobacter sakazakii) from food, animal feed and environmental samples in areas of food production and handling.
According to ISO 22964:2017, CCI is used in conjunction with Cronobacter Selective Broth (CSB).

METHOD PRINCIPLE
Tryptic digest of casein provides amino acids, nitrogen, carbon, minerals and vitamins essential for bacterial growth. Yeast extract is a source of vitamins, particularly of B-group. Sodium chloride maintains the osmotic balance of the medium. 5-Bromo-4-Chloro-3-Indoxyl-α-D-Glucopyranoside is the chromogenic substrate. Sodium desoxycholate is the selective agent inhibiting the growth of Gram positive competitors. Ammonium iron citrate and sodium thiosulfate are included to differentiate hydrogen sulphide-producing Enterobacteriaceae. Agar is the solidifying agent.

TYPICAL FORMULA (g/l)

PREPARATION
Dehydrated medium : Suspend 32.4 g of the powder in 1 liter of distilled or deionized water. Mix well.
Heat to boil shaking frequently until completely dissolved. Sterilize in autoclave at 121°C for 15 minutes. Cool to 45-50°C and pour into Petri dishes.

TEST PROCEDURE

1. To prepare the initial suspension, add 10 g or 10 ml of the test sample to 90 ml of Buffered Peptone Water (ref. 414030).
2. Incubate the inoculated pre-enrichment medium at 36 ± 2°C for 18 ± 2 h.
3. Mix well and transfer 0.1 ml of the pre-enrichment culture to a tube of CSB (ref. 24483).
4. Incubate the inoculated enrichment medium at 41.5°C for 24 ± 2 h.
5. Select tubes which appear yellow and use a 10 μl loop to streak the enrichment culture onto plates of CCI.
6. Incubate the plates at 41.5°C for 24 ± 2 h.

INTERPRETING RESULTS
After incubation observe the color of the colonies and interpret the results as indicated in the ID table. For confirmation, select 1 to 5 typical colonies and streak onto a non-selective agar, such as Tryptc Soy Agar (ref. 10037), in order to achieve well isolated colonies. Incubate at 36 ± 2°C for 21 ± 3 h. Refer to ISO 22964 for more details.

ID Table.

APPEARANCE
Dehydrated medium: free-flowing, homogeneous, yellowish.
Prepared medium: slightly opalescent, light amber.

STORAGE
The powder is very hygroscopic, store the powder at 10-30°C, in a dry environment, in its original container tightly closed. Store plates at 2-8°C away from light. Do not use the product beyond its expiry date on the label or if product shows any evidence of contamination or any sign of deterioration.

SHELF LIFE
Dehydrated medium: 4 years.
Ready-to-use plates: 6 months.

QUALITY CONTROL
Plates are inoculated with the microbial strains indicated in the QC table.
Inoculum for productivity: 50-100 CFU.
Inoculum for selectivity: 10⁴-10⁶ CFU.
Inoculum for specificity: 10³-10⁴ CFU.
Incubation conditions: aerobically at 41.5 ± 1°C for 24 ± 2 h.

QC Table.

WARNING AND PRECAUTIONS
The product does not contain hazardous substances in concentrations exceeding the limits set by current legislation and therefore is not classified as dangerous. It is nevertheless recommended to consult the safety data sheet for its correct use. The product is intended for professional use only and must be used by properly trained operators.

DISPOSAL OF WASTE
Disposal of waste must be carried out according to national and local regulations in force.

PRESENTATION

BIBLIOGRAPHY

1. ISO 22964:2017. Microbiology of the food chain − Horizontal method for the detection of Cronobacter spp.
2. EN ISO 11133:2014. Microbiology of food, animal feed and water − Preparation, production, storage and performance testing of culture media.
3. Guillame-Gentil O., Sonnard V., Kandahai M.C., Mauragg J.D., and Jootsen H.A. (2005) A simple Rapid Cultural Method for Detection of Enterobacter sakazakii in environmental samples. J Food Prot. 68 (1): 64-69.

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