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ENTEROBACTER SAKAZAKII ISOLATION AGAR

CODE: CM1134

A chromogenic medium for the detection and enumeration of Cronobacter (formerly E. sakazakii) in milk and milk samples as described in ISO TS/22964:20061. To be used in conjunction with mLST/vancomycin medium (CM1133 & SR0247E)

Typical Formula*

gm/litre

Pancreatic digest of casein†

7.0

Yeast extract

3.0

Sodium chloride

5.0

Sodium deoxycholate††

0.6

5-Bromo-4-chloro-3-indolyl α-D-glucopyranoside

0.15

Crystal violet

0.002

Agar

12.0

pH 7.0 ± 0.2 @ 25°C

 

*Adjusted as required to meet performance standards
†Pancreatic digest of casein = pancreatic peptone of casein
††Sodium deoxycholate = Sodium desoxycholate

Directions
Suspend 27.75g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilize by autoclaving at 121°C for 15 minutes. Cool to approximately 50°C. Mix well and pour into sterile Petri dishes.

Description
Cronobacter (formerly Enterobacter sakazakii) are Gram-negative rod-shaped members of the Family Enterobacteriaceae that have been implicated in outbreaks of disease in premature infants (neonates), causing sepsis, meningitis and necrotising enterocolitis. Neurological damage can be permanent, and the death rate is reported to be as high as 40-80%1.

Cronobacter spp. have been isolated at low levels from powdered infant formulae, and the organisms’ high tolerance to desiccation provides a competitive advantage in the dry environments of milk powder factories, increasing the risk of product contamination2.

In response to concerns over this pathogen, a collaborative technical specification for the detection of Cronobacter in milk and milk products was jointly issued by ISO and the IDF, as ISO/TS22964:20063. This recommended pre-enrichment in BPW, selective enrichment in mLST/vancomycin broth and plating on Enterobacter sakazakii Isolation Agar. Positive colonies are confirmed on the basis of sugar utilisation, decarboxylase activity, as well as yellow pigmentation.

Unlike most other members of the Enterobacteriaceae, Cronobacter possess the enzyme α-glucosidase, which is used as a diagnostic feature in chromogenic medium. Enterobacter sakazakii Isolation Agar contains a substrate for this enzyme, 5-bromo-4-chloro-3-indolyl α-D-glucopyranoside (X-α-gluc). Cronobacter hydrolyse this colourless chromogen to produce characteristic blue-green colonies for identification on the plate. Sodium desoxycholate and crystal violet are also incorporated in the medium to inhibit the growth of Gram-positive organisms.

The European Union has directly specified limits for the levels of Cronobacter in dried infant formulae, dried dietary foods for special medical purposes and food intended for infants below six months of age, in Commission Regulation no 2073/20054. This regulation states for products placed on the market, during their shelf life, 30 samples of 10g have to be tested, and the food producer has to demonstrate absence of Cronobacter in each of these samples, using ISO/TS 22964:20063.

Technique
Refer to the relevant standard method for detailed instructions. The following method is a summary of ISO/TS 22964:20063

  1. Prepare test sample in accordance with ISO 68875, ISO 82616 or the appropriate standard
  2. Homogenise x g of sample in 9 times x ml of Buffered Peptone Water ISO (CM1049)
  3. Incubate samples at 37°C ± 1°C for 18h ± 2h
  4. Transfer 0.1ml of enrichment into 10ml of mLST/vancomycin broth (CM1133 & SR0247)
  5. Incubate at 44°C ± 0.5°C for 24h ± 2h
  6. Using a 10µl loop, streak onto the surface of the Enterobacter sakazakii Isolation Agar plate (CM1134), invert and incubate the dishes at 44°C ± 1°C for 24h ± 2h
  7. Select 1-5 blue-green colonies for confirmation
  8. Streak onto Tryptone Soya Agar (CM0131) plates, incubate at 37°C ± 1°C for 46 ± 2 hours and check for yellow coloration
  9. Perform required biochemical confirmation tests

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
The prepared medium is best used freshly prepared, but may be suitable for longer storage following in-house validation.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Purple coloured gel

Quality Control

Positive Controls

Expected results

Cronobacter sakazakii ATCC® 12868

1-2mm blue-green colonies

Cronobacter sakazakii ATCC® 29004

1-2mm blue-green colonies

Cronobacter muytjensii ATCC® 51329*

1-2mm blue-green colonies

Negative Controls

 

Escherichia coli ATCC® 25922*

0.25-1.5mm purple colonies

Staphylococcus aureus ATCC® 25923*

Inhibited or no growth

*This organism is available as a Culti-Loop®

References

  1. Mullane et al. 2007. Minerva Pediatr. 59. 137-148
  2. Lai. 2001. Medicine. 80. 113-122
  3. Anonymous. 2006. Milk and milk products-detection of Enterobacter sakazakii Technical specification ISO/TS22964:2006(E)
  4. Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs
  5. ISO 6887-1:1999 Microbiology of food and animal feeding stuffs -- Preparation of test samples, initial suspension and decimal dilutions for microbiological examination.
  6. ISO 8261:2001 Milk and milk products -- General guidance for the preparation of test samples, initial suspensions and decimal dilutions for microbiological examination
 
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