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Dehydrated Culture Media

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mLST / VANCOMYCIN MEDIUM

For the selective enrichment of Cronobacter (formerly E. sakazakii) from milk and milk samples as described in ISO TS/22964:20061. To be used in conjunction with Enterobacter sakazakii Isolation Agar (CM1134)

MODIFIED LAURYL SULPHATE BROTH (mLST)

CODE: CM1133

Typical Formula*

gm/litre

Sodium Chloride

34.0

Enzymatic digest of animal and plant tissue

20.0

Lactose

5.0

Potassium dihydrogen phosphate 

2.75

Dipotassium hydrogen phosphate

2.75

Sodium lauryl sulphate

0.1

pH 6.8 ± 0.2 @ 25°C

 
*Adjusted as required to meet performance standards

VANCOMYCIN SUPPLEMENT (5mg)

CODE:  SR0247E   

Vial contents: (each vial sufficient for 500ml of medium)
per vial
per litre
Vancomycin
5mg
10mg

Directions
Dissolve 32.3g in 500ml of distilled water. Sterilize by autoclaving at 121°C for 15 minutes. Cool to approximately 50°C, and aseptically add the contents of 1 vial of Vancomycin Supplement (5mg) (SR0247E) reconstituted as directed. Mix well and aseptically dispense into sterile containers.

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, followed by selective enrichment in mLST/vancomycin (mLSTv) broth and plating on Enterobacter sakazakii Isolation Agar (CM1134). Positive colonies are confirmed on the basis of sugar utilisation, decarboxylase activity, as well as yellow pigmentation.

MLST broth was originally described by Guillaume-Gentil4 et al. for the selective enrichment of Cronobacter. The high salt concentration of the medium (34g/l) restricts the growth of other members of the Enterobacteriaceae, and the combination of vancomycin and sodium lauryl sulphate inhibits the growth of Gram-positive organisms commonly isolated in production environments. A careful blend of peptones and lactose ensures Cronobacter remain viable in the presence of these selective agents

The European Union has 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 2073/20055.  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 68876, ISO 82617 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 mLSTv 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 plates at 44°C ± 1°C for 24h ± 2h
  7. Select 1-5 blue/green colonies for confirmation
  8. Streak onto Tryptone Soya Agar plates, and incubate at 37°C ± 1°C for 18h to 24h and check for yellow colouration
  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
Where possible the complete medium should be prepared on the day of testing and stored for no longer than 1 day at 0-5ºC

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

Quality Control

Positive Controls

Expected results

Cronobacter sakazakii ATCC® 12868

Turbid growth

Cronobacter sakazakii ATCC® 290041

Turbid growth

Cronobacter muytjensii ATCC® 51329*

Turbid growth

Negative Controls

 

Escherichia coli  ATCC® 25922*

Inhibited or no growth

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. Guillaume-Gentil et al. 2005. J. Food. Prot. 68. 64-69
  5. Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs
  6. ISO 6887-1:1999 Microbiology of food and animal feeding stuffs -- Preparation of test samples, initial suspension and decimal dilutions for microbiological examination.
  7. 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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