SS Agar is a selective and differential medium for the isolation, identification and differentiation of Salmonella and some Shigella species from clinical and non-clinical samples, suspected foodstuffs and for microbial limit test. SS Agar is a modification of the Desoxycholate Citrate Agar. Salmonella Shigella (SS) Agar is highly selective for Salmonella species, but is inhibitory to some strains of Shigella. Therefore it should not be used for the primary isolation of Shigella, instead an alternate product should be used.
SS Agar is a moderately selective medium in which gram-positive bacteria and coliforms are selectively inhibited by bile salts, brilliant green and sodium citrate. Certain enteric bacteria can reduce Sodium thiosulphate to sulphite and H2S gas. The Production of H2S gas is detected as an insoluble black precipitate of ferrous sulphide in the centre of the colonies, which is formed upon reaction of H2S with ferric ions or ferric citrate. On fermentation of lactose by few lactose-fermenting normal intestinal flora, acid is produced which is indicated by change of colour from yellow to red by the pH indicator-neutral red. Thus these organisms grow as red pigmented colonies.
Composition of Salmonella Shigella Agar
Ingredients Gms / Litre
Beef Extract 5.00
Enzymatic Digest of Casein 2.50
Enzymatic Digest of Animal Tissue 2.50
Bile Salts 8.50
Sodium Citrate 8.50
Sodium Thiosulfate 8.50
Ferric Citrate 1.00
Brilliant Green 0.00033
Neutral Red 0.025
Distilled Water 1000 ml
pH ( at 25°C) 7.0 ± 0.2
- Lactose:source of fermentable carbohydrate
- Beef extract, proteose peptone:supplies nitrogen, vitamins, and amino acids
- Sodium thiosulphate &Sodium citrate: act as a selective agents by providing an alkaline pH to inhibit the growth of Gram-positive organisms and coliforms
- Ferric citrate: Sodium Thiosulfate is also a source sulfur
- Bile salts : The bile salts inhibit growth of gram-positive microorganisms
- Brilliant Green/Neutral Red: pH indicator
- Agar:Solidifying agent.
Colour and Clarity of prepared medium
Reddish orange coloured clear to slightly opalescent gel forms in Petri plates.
Reaction of 6.0% w/v aqueous solution at 25°C. pH : 7.0±0.2
Cultural characteristics observed after an incubation at 35 – 37°C for 18 – 24 hours
Uses of Salmonella Shigella Agar
- It is used as a selective and differential medium for the isolation of Salmonella and some Shigellaspecies from clinical and non-clinical specimens.
- This medium is not recommended for the primary isolation of Shigella.
- It is used to differentiate lactose and non-lactose-fermenters from clinical specimens, suspected foods, and other such samples.
- The high selectivity of SS Agar allows the use of large inoculum directly from faeces, rectal swabs or other materials suspected of containing pathogenic enteric bacilli.
Preparation of Salmonella Shigella Agar
- Dissolve 60.0 grams of SS Agar in 1ltr dH20
- Heat to boiling to dissolve the medium completely.
- Do not autoclave.
- Mix well and pour into sterile Petri plates.
Salmonella Shigella Agar should appear clear, and red-orange in color.
Storage and Shelf Life
Store below 30°C away from direct sun light and the prepared medium should be stored at 2-8°C
- Allow medium to reach room temperature.
- using a heavy inoculum, perform a four-quadrant streak to obtain well-isolated colonies.
- Incubate aerobically at 35°C.
- Examine after 18-24 hours. Re-incubate plates for an additional 24 hours to detect late lactose fermenters.
Result Interpretation on Salmonella Shigella Agar
- Salmonellaare lactose non-fermenters, but produce hydrogen sulfide (H2S) gas. They produce translucent bacterial colonies with black centers.
- Shigellaspecies are lactose non-fermenters and do not produce hydrogen sulfide gas, so produce transparent colonies
- Coliform bacteria are patially inhibited on Salmonella ShigellaAgar but may show growth after prolonged incubation
- coli ferment the lactose present in the media and produces pink to red colonies. They do not produce any hydrogen sulfide.
- Enterobacter andKlebsiella appears larger than coli and produce mucoid, pale, opaque cream to pink colonies
- Citrobacter and Proteus species form colonies with gray or black centers.
Limitations of Salmonella Shigella Agar
- It is recommended that serological biochemical, molecular, or mass spectrometry testing be performed on colonies isolated from pure culture in order to complete identification.
- The incorporation of brilliant green into this medium makes it highly selective, and has been shown to inhibit the growth of some Shigella
- The bile salts may get precipitated over time and appear as small spider-like puff balls within the medium and do not affect the performance of the medium.
- Some strains of Shigella, such as sonnei and dysenteriae serovar 1, may ferment lactose relatively slowly, and colonies change to lactose-fermenting after incubation of 2 or more days.
- A few non-pathogenic organisms may grow on Salmonella Shigella