ITC Supplement

Description

Enzymatic casein digest provides nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group, essential for bacterial growth. Magnesium chloride and malachite green make the broth highly selective. Irgasan inhibits Gram-positive bacteria, ticarcillin has a bactericidal action on Gram-negative and Gram-positive bacteria and potassium chlorate has a disinfecting property.

Technique

Aseptically reconstitute 1 vial with 10 mL of sterile distilled water. Mix gently until complete dissolution and aseptically add to 500 mL of Irgasan Ticarcillin and Potassium Chlorate Broth (ITC) ISO (LC-1361), autoclaved and cooled to 50 °C. Mix well and distribute into sterile containers.

Instructions for use

For the detection of Yersinia enterocolitica according to ISO 10273:

• To obtain the initial suspension, mix 25 g of the sample with 225 mL of PSB (LC-1298) preheated to room temperature.
• Prepare the suspension in Irgasan Ticarcillin and Potassium Chlorate Broth (ITC) (LC-1361) by transferring 10 mL of PSB into 90 mL of ITC medium.
• Using the initial suspension in PSB, divide 1 mL between 2-4 CIN Agar (LC-1126) plates and spread it on the plate surface using a spreader. Incubate at 30 °C for 24 ± 2 hours.
• Incubate both suspensions at 25 °C for 44 ± 4 hours.
• Transfer 0.5 mL of each suspension into 4.5 mL of KOH solution (separately) prepared the day before. Inoculate both solutions in CIN Agar plates.
• Incubate the plates in an inverted position at 30 °C for 24 ± 2 hours.
• Verify the morphology of the colony as presumptive pathogenic Y. enterocolitica by successive culturing on selective plates. Typical colonies of Y. enterocolitica will appear colorless, with dark red centers, like a bull's eye, surrounded by a transparent border.
• Confirm the presence of pathogenic species of Y. enterocolitica by biochemical or molecular confirmation tests.

Reconstitute the original freeze-dried vial by adding 10 mL of sterile distilled water.

Note: each vial is sufficient to supplement 500 mL of ITC Broth.

Physical/chemical control

Microbiological control

Add 1 vial to 500 mL of medium base. DO NOT HEAT once supplemented.

Inoculate: practical range 100 ± 20 CFU, min. 50 CFU (productivity) / 10⁴-10⁶ (selectivity).

Aerobiosis. Incubation at 25 ± 1 °C, reading at 44 ± 4 h.

Microbiological control according to ISO 11133:2014/Amd 1:2018.

Sterility control

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

• ISO 10273 Standard. (2003) Microbiology of food and animal feeding stuffs - Horizontal methods for the detection of presumptive pathogenic Yersinia enterocolitica.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• American Public Health Association Compendium of Methods for the microbiological Examination of Foods.
• Schiemann, D.A. Synthesis of a selective agar medium for Yersinia enterocolitica. Canad. J. Microbiol. 25: 1298-1304.