Iron Sulfite Agar ISO

Enzymatic digest of casein and soy peptone provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is source of vitamins, particularly the B-group, essential for bacterial growth. Ferric citrate and sodium disulfite are H₂S indicators: Clostridium perfringens reduces the sulfite to sulfide, which in turn reacts with the iron and forms a black iron sulfide precipitate, seen as black colonies. Bacteriological agar is the solidifying agent.

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 40 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute or until complete dissolution. Distribute in flasks and sterilize in autoclave at 121 °C for 15 minutes.

According to ISO 15213

Enumeration of sulfite-reducing bacteria growing under anaerobic conditions according to ISO 15213:

1. Take plates or tubes to prepare the Iron Sulfite medium.
2. If plates are used, transfer to each dish 1 mL of the first decimal dilution (10⁻¹) of the test sample if the product is liquid, or 1 mL of the first decimal dilution of the initial suspension (10⁻²) in the case of other products.
3. Pour into each Petri dish approximately 15 mL of Iron Sulfite Agar.
4. Mix it carefully and allow the medium to solidify.
5. After the medium has solidified, pour 5 mL to 10 mL of the same medium into the dish as an overlay.
6. If tubes are used, inoculate 1 mL from each dilution into each of two tubes of medium.
7. Mix gently and leave the medium to solidify.
8. After the medium has solidified, pour 2 to 3 mL of the same medium into each tube as an overlay.
9. Incubate the plates or tubes in anaerobic jars at 37 ± 1 °C for 24-48 hours.
10. If thermophilic bacteria are suspected, incubate at 50 ± 1 °C.

According to ISO 6461

Detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) by the membrane filtration method according to ISO 6461:

1. Apply heat to the sample for a period of time sufficient to destroy the vegetative bacteria (75 ± 5 °C for 15 minutes).
2. Filter the water sample through a membrane filter.
3. Place the filter on the Iron Sulfite Agar.
4. Incubate at 37 ± 1 °C for 20 ± 4 hours and 44 ± 4 hours in anaerobic conditions.
5. Count all black colonies.

• ISO 15213. Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of sulfite-reducing bacteria growing under anaerobic conditions.