Trypticasein Soy Broth Modified with Novobiocin (mTSB) ISO

Casein peptone and soy peptone provide nitrogen, vitamins, minerals and amino acids essential for growth. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Dipotassium phosphate acts as a buffer system. Glucose is the fermentable carbohydrate providing carbon and energy. Bile salts and novobiocin are inhibitors of Gram-positive organisms.

ISO 16654 recommends preparing the initial suspension and adding a test portion to the 41.5 °C pre-warmed broth to obtain a ratio of test portion to mTSB + sample of 1/10 (mass to volume, or volume to volume). Incubate for 6 hours then a further 12 hours to 18 hours at 41.5 °C.

Preparation

Suspend 33 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into tubes and sterilize in autoclave at 121 °C for 15 minutes.

According to ISO 16654

For the detection of Escherichia coli O157 according to ISO 16654:

1. Prepare the initial suspension by adding the test portion to Trypticasein Soy Broth Modified with Novobiocin prewarmed to 41.5 °C to obtain a ratio of 1/10.
2. Separate and concentrate the microorganisms by immunogenic particles coated with antibodies to E. coli O157.
3. Incubate for 6 hours then a further 12 hours to 18 hours at 41.5 °C.
4. Subculture the immunomagnetic particles with the bacteria adhered on MacConkey Agar with Sorbitol (LC-1099) and a second selective isolation agar chosen by the laboratory. The optimal incubation temperature for E. coli O157 is 37 ± 1 °C for 18-24 hours.
5. Confirm by indole production in Tryptophan Culture Broth (LC-1237) and agglutination with the anti-E. coli O157 serum.

• ISO 16654:2001 Microbiology of food and animal feed stuffs. Horizontal method for the detection of E. coli O157.
• PHLS Standard Methods F17 & W16: Detection of E. coli O157.
• FDA/BAM 8th Edition (Rev. A) (1998) Chapter 4: 4.20-4.26.