Chromogenic medium for detection of Cronobacter spp (formerly E. sakazakii), according to ISO 22964

DESCRIPTION
Enterobacter (Cronobacter) sakazakii Isolation Agar is a chromogenic medium used for the selective isolation of Cronobacter spp (formerly E. sakazakii) in infant milk products, according to ISO 22964.

METHOD PRINCIPLE
Pancreatic digest of casein provides amino acids, nitrogen, carbon, vitamins and minerals for organisms 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-indolyl α-D-glucopyranoside is the chromogenic substrate. Sodium desoxycholate and crystal violet inhibit Gram-positive bacteria. Agar is the solidifying agent.

TYPICAL FORMULA (g/l)

PREPARATION
Dehydrated medium: Suspend 31.0 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.

TEST PROCEDURE
ISO 22964 recommends the following procedure:

1. Homogenize the sample diluted 1:10 in Buffered Peptone Water (ref. 24099) and incubate at 37 ± 1°C for 18 ± 2 hours.
2. Transfer 0.1 ml of the enrichment culture into 10 ml of Lauryl Sulfate Tryptose Broth Modified (mLST) (ref. 24452) and incubate at 44 ± 0.5°C for 24 ± 2 hours.
3. Streak a loopful (10 μl) onto the surface of an Enterobacter (Cronobacter) Sakazakii Isolation Agar plate and incubate aerobically at 44 ± 1°C for 24 ± 2 hours.
   Alternatively, samples can be directly streaking onto the plate omitting pre-enrichment and enrichment steps.

INTERPRETING RESULTS
After incubation observe the color of the colonies and interpret the results as indicated in the ID table.
Confirm typical colonies with proper biochemical tests.

ID Table.

APPEARANCE
Dehydrated medium: free-flowing, homogeneous, beige.
Prepared medium: slightly opalescent, purplish blue.

STORAGE
The powder is very hygroscopic, store the powder at 10-30°C, in a dry environment, in its original container tightly closed. Store prepared 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 44 ± 1°C for 24 ± 2hours.

QC Table

PRESENTATION

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.

BIBLIOGRAPHY

1. ISO 22964:2006. Milk and milk products detection of Enterobacter sakazakii.
2. Guillame-Gentil O., Sonnard V., Kandahai M.C., Mauragg J.D., and Jootsen H.A. A simple Rapid Cultural Method for Detection of Enterobacter sakazakii in environmental samples. J Food Prot. 68 (1), 2005, 64-69.

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