Muller Kaufmann Broth Base with Brilliant Green and Novobiocin (MKTTN) ISO

Beef extract and casein peptone provide nitrogen, vitamins, minerals and amino acids essential for growth. Calcium carbonate is a neutralizer which absorbs toxic metabolites. Bile salts, brilliant green and novobiocin inhibit organisms other than Salmonella. Selectivity is also obtained by both sodium thiosulfate and tetrathionate, suppressing coliforms. Tetrathionate is formed in the medium with the addition of the iodine and potassium iodide solution. Organisms containing the enzyme tetrathionate reductase will thrive in this medium. Sodium chloride supplies essential electrolytes for transport and osmotic balance.

Preparation

Suspend 89.53 grams of the medium in one litre of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. AVOID OVERHEATING. DO NOT AUTOCLAVE. Cool to 45-50 °C. Aseptically add 20 mL of an iodine and potassium iodide solution (20 g of iodine and 25 g of potassium iodide in 100 mL of sterile distilled water). Homogenize gently and dispense into sterile containers.

According to ISO 6579

For the detection of Salmonella spp. in food, animal feed, animal faeces and environmental samples according to ISO 6579:

1. Pre-enrichment in non-selective liquid medium: inoculate the Buffered Peptone Water (LC-1402) with the sample or dilutions, and incubate at 34-38 °C for 18 ± 2 h.
2. Enrichment in/on selective media: inoculate, with the culture obtained in the pre-enrichment stage, the Rappaport Soy Broth (Vassiliadis) (LC-1174) or the Modified Semisolid Rappaport Vassiliadis medium (MSRV) (LC-1376), and the MKTTN Broth. The Rappaport Soy Broth and the Modified Semisolid Rappaport medium are incubated at 41.5 °C for 24 ± 3 h, and the MKTTN Broth at 34-38 °C for 24 ± 3 h.
3. Plating out on selective solid media: from the selective enriched cultures, inoculate two selective isolation agars: XLD agar (LC-1274) and any other selective medium complementary to XLD agar (Salmonella Chromogenic Agar (LC-1122), Brilliant Green Agar (LC-1143), Bismuth Sulfite Agar (LC-1011), DCLS Agar (LC-1045), Desoxycholate Citrate Agar (LC-1067), Hektoen Enteric Agar (LC-1030), Salmonella Shigella Agar (LC-1064) and XLT4 Agar (LC-1159)). Incubate the XLD plates inverted at 34-38 °C for 24 ± 3 h. Incubate the second selective medium in accordance with the manufacturer's instructions.
4. Confirmation: subculture colonies of presumptive Salmonella and confirm their identity by biochemical and serological tests.

Note: according to Annex D of ISO 6579-1:2017, for the detection of enterica subspecies enterica serovars Typhi and Paratyphi, Selenite Cystine Broth (LC-1220) should be added as a selective enrichment medium and Bismuth Sulfite Agar (Wilson Blair) should be selected as a second selective medium (LC-1011).

According to ISO 19250

For the detection of Salmonella spp. in water samples according to ISO 19250:

1. Pre-enrichment in non-selective medium: inoculate the Buffered Peptone Water (LC-1402) with the sample or dilutions, and incubate at 34-38 °C for 18 ± 2 h.
2. Enrichment in selective media: inoculate, with the culture obtained in the pre-enrichment stage, the Rappaport Soy Broth (Vassiliadis) (LC-1174) and the MKTTN Broth. The Rappaport Soy Broth is incubated at 41.5 ± 1 °C and the MKTTN Broth at 34-38 °C, both of them for 24 ± 3 hours.
3. Plating out on selective solid media: from the selective enriched cultures, inoculate two selective isolation agars: XLD agar (LC-1274) and any other selective medium complementary to XLD agar (for instance, Brilliant Green Agar (LC-1143) or Bismuth Sulfite Agar (LC-1011)). Incubate the XLD plates inverted at 34-38 °C for 24 ± 3 hours. Incubate the second selective medium in accordance with the manufacturer's instructions.
4. Confirmation: subculture colonies of presumptive Salmonella and confirm their identity by biochemical and serological tests.

• ISO 6579 Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp.
• ISO 6579 Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. Amendment 1: Broader range of incubation temperatures, amendment to the status of Annex D, and correction of the composition of MSRV and SC.
• Kauffmann, F. 1935. Weitere erfahrungen mit dem kombinierten anreicherungsverfahren fur Salmonella bazillen. Ztschr. F. Hyg. 117: 26-32.
• ISO 19250 Water quality - Detection of Salmonella spp.