Iron Sulphite Agar, Granulated

Principle And Interpretation

Iron Sulphite Agar is a modification of Cameron Sulphite Agar developed by the National Canners Association of America (1). It was shown by Beerens (2) that 0.1% sulphite concentration in the original formula was inhibitory to some strains of Clostridium sporogenes. This observation was later confirmed by Mossel et al (3), who consequently showed that 0.05% sulphite concentration was not inhibitory to the organisms. Most clostridia have sulfite reductase in their cytoplasm but they are unable to expel them to the exterior. So when H2S is produced from sulfite, the colony becomes dark due to the formation of precipitates of iron sulfide from citrate.

Tryptone provides nitrogen and other nutrients necessary to support bacterial growth. Sulphite-reducing bacteria usually produce black colonies as a result of the reduction of sulphite to sulphide, which reacts with the iron (III) salt.

For the detection of organisms causing sulphide spoilage, two methods can be followed:

1. Deep-Shake Culture Method: Dispense the medium in 10 ml amounts in tubes. Inoculate the sample when the medium is at about 50°C. Allow to set and incubate at 55°C for 24-48 hours.
   Typical thermophilic species -Desulfotomaculum nigrificans, produces distinct black spherical colonies in the depth of the medium.
2. Attenborough and Scarr (4) Method: In this method, diluted samples of sugar or any other food are filtered through membrane filters.

These filters are then rolled up and placed in tubes containing just sufficient Iron Sulphite Agar (at 50°C) to cover them. The medium is allowed to set and then incubated at 55-56°C for 24-48 hours. After incubation, the number of black colonies on the membrane filter is counted. Confirmation tests are further carried out to identify the organism growing in the medium. This membrane filter technique is quicker, of comparable accuracy and permits the examination of larger samples. The blackening reaction is only presumptive evidence of clostridial growth. Confirmation test must be carried out for identification. There are many gram-negative bacteria that are able to reduce sulfite with iron sulfide production in this medium, but in these cases the enzymes are extra cellular and the entire medium becomes dark, rendering their enumeration impossible.