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Dehydrated Culture Media

CHROMOGENIC CRONOBACTER ISOLATION AGAR (CCI)

A new chromogenic medium for the detection of Cronobacter (formerly E. sakazakii) from food and environmental samples. To be used in conjunction with Cronobacter Screening Broth (CM1121)

CODE: CM1122

Typical Formula*

gm/litre

Yeast extract

3.0

Sodium chloride

5.0

Tryptone

7.0

Sodium thiosulphate

1.0

Sodium desoxycholate

0.25

Ferric ammonium citrate

1.0

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

0.15

Agar

13.2

pH 7.3 ± 0.2 @ 25°C

 
*Adjusted as required to meet performance standards

Directions
Suspend 30.6g 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.

The ongoing development of a new horizontal CEN-ISO standard method for the detection of Cronobacter spp by the ISO/TC 34, SC 9Ad hoc group for E. sakazakii, addresses issues such as the scope of the method, reliability of identification criteria and improvements to the media1. In anticipation of these future needs, Oxoid has launched new Cronobacter Screening Broth (CM1121) and Chromogenic Cronobacter Isolation Agar (CM1122).

Chromogenic Cronobacter Isolation (CCI) agar is an optimisation of the original Brilliance™ Enterobacter Sakazakii Agar (DFI), developed by Iversen et al. in 20043. The concentration of the X-α-glucoside chromogen was increased to improve the blue-green colouration of Cronobacter strains with weak glucosidase activity. Sodium thiosulphate and ammonium iron (III) citrate are included to differentiate hydrogen sulphide-producing Enterobacteriaceae, which are coloured grey/brown, and sodium deoxycholate is added to inhibit the growth of Gram positive competitors.

Technique

  1. Prepare test sample in accordance with ISO 68874, ISO 82615 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 CSB, and incubate at 41.5°C ± 1°C for 24h ± 2h
  5. If broth is purple, the result is negative, and no further work is required. If broth is yellow proceed to step 6.
  6. Using a 10µl loop, streak onto the surface of the CCI Agar plate
  7. Invert and incubate the plates at 44°C ± 1°C for 24h ± 2h
  8. Select 1-5 blue/green colonies for confirmation
  9. Streak onto Tryptone Soya Agar plates, and incubate at 37°C ± 1°C for 18h ± 2h
  10. 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
Store the prepared medium at 2-8°C

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

Quality Control

Positive Controls

Expected results

Cronobacter sakazakii ATCC® 12868

1-2mm blue-green colonies

Cronobacter sakazakii ATCC® 290041

1-2mm blue-green colonies

Cronobacter muytjensii ATCC® 51329*

 1-2mm blue-green colonies

Negative Controls

 

Escherichia coli  ATCC® 25922*

0.25-1.5mm straw 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. Iversen et al. 2008. Appl. Environ. Microbiol. 74, 2550-2552
  4. ISO 6887-1:1999 Microbiology of food and animal feeding stuffs -- Preparation of test samples, initial suspension and decimal dilutions for microbiological examination.
  5. 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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