Salmonella Shigella Agar (SS Agar) Principle, Composition, Application

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Salmonella Shigella Agar (SS Agar)

  • Salmonella Shigella (SS) Agar can be moderately specific and a differing medium used for the cultivation, isolation as well as differentiation Salmonella spp. and a few varieties that belong to Shigella spp.
  • The SS Agar is a variation of Deoxycholate Citrate Agar.
  • It is recommended to test the clinical specimens and food samples to detect evidence of Salmonella Spp. and a few Shigella species.
  • SS Agar is a moderately selective medium that Gram-positive bacteria are slowed down by bile salts and a brilliant green, and sodium citrate. Salmonella Shigella (SS) Agar extremely specific for Salmonella species, however it is ineffective for certain varieties of Shigella.
  • It was developed to aid in the differentiation of lactose and non-lactose-fermenters from clinical specimens, suspected foods, and other such samples.

Principle of SS Agar (Salmonella Shigella Agar)

  • SS Agar is recommended as a differential and selective medium for the identification of Salmonella as well as Shigella strains from pathological samples and suspected food products and also for the testing the microbial limit. 
  • SS Agar is a moderately selective medium, where Gram-positive bacteria are suppressed by Bile salts, brilliant blue as well as sodium citrate. 
  • The digestive product of the animal tissues, beef extract provides essential nutrients for growth. Lactose is a fermentable carbohydrate. 
  • The brilliant green color, the bile and the thiosulphate inhibit selectively gram-positive and bacteria. 
  • The sodium thiosulphate in the body is reduced by specific species of enteric bacteria to H2S gas as well as sulphite. the reductive enzyme reaction is explained by thiosulphate reductase. 
  • The gas produced by H2S is recognized as an insoluble, dark precipitate that is ferrous Sulphide that is formed by the reaction of H2S with ferric ions, or ferric citrate, which is visible in the middle of colonies. 
  • The high sensitivity of Salmonella Shigella agar allows for the application of huge doses of inocula directly from the rectal swabs, faeces and other substances suspected to contain pathogenic enteric bacteria. 
  • In the process of fermentation of lactose, through a small amount of lactose-fermenting flora of normal intestinal flora acid is generated, and is evident by the change in colour from red to yellow by the pH indicator, which is neutral red. This is why these organisms form red-colored colonies. 
  • The non-fermenting Lactose species form transparent colonies that are colourless and with black centers.
  • The growth of Salmonella species is unimpeded and is seen as colorless colonies with black centre resultant from the production of H2S. 
  • Shigella species can also develop as colorless colonies that don’t produce H2S. 
  • It is recommended to place plates with less inhibiting media in parallel with SS Agar, such as Hektoen Enteric Agar (M467) or Deoxycholate Citrate (M065) to make it easier to isolate for Shigella varieties.

SS Agar Composition

Bile salts no.38.5
Sodium citrate8.5
Sodium thiosulfate8.5
Beef extract5.0
Proteose peptone5.0
Ferric Citrate1.0
Brilliant green0.00033
Neutral red0.025

Final pH 7.0 +/- 0.2 at 25ºC.

Step by Step Preparation of SS Agar

  1. You should suspend 60 grams of the substance in 1 liter deionized or distillate water.
  2. Mix the sample thoroughly.
  3. Stir frequently and heat to boil for about one minute.
  4. Do not autoclave the media.
  5. Pour the sample into plates.
  6. Let the agar set and put it within the fridge (avoid chilling). Culture media prepared can be stored for a minimum of one week in the refrigerator.

Culturing the sample on SS Agar

  1. Let the plates be at room temperature, and allow the surface of the agar to dry prior to inoculating.
  2. Inoculate thoroughly to streak your sample as quickly as is possible following collection.
  3. If the sample to cultivated has been placed on a sample use the swab to roll it over only a small portion of the surface of the agar.
  4. Streak for isolation using an sterilized loop.
  5. Incubate the plates in aerobic mode at 35-37 degrees C for 18-24 hours.
  6. Examine colonial morphology.

Result Interpretation on SS Agar

  • Lactose fermenter: The medium will turn red due to the acidic pH. e.g. Escherichia coli, Klebsiella pneumoniae gives red colonies.
  • Non-Lactose fermenter: Salmonella, Shigella, and other non-lactose fermenters appear as transparent or translucent colorless colonies. Colonies of Salmonella spp. may appear with or without black centers (depending on the species isolated).
ShigellaClear, colorless, transparent
Escherichia coliSmall, pink to red
Enterobacter, KlebsiellaLarger than E.coli, mucoid, pale, opaque cream to pink
SalmonellaColorless, transparent, with a black center if H2S is produced
ProteusColorless, with black center
Salmonella Shigella (SS) Agar result
Salmonella Shigella (SS) Agar result

Application of SS Agar

  • It can be used as a discriminating and selective medium to isolate Salmonella and a few Shigella species from non-clinical and clinical samples.
  • This medium is not suggested as a primary isolate of Shigella.
  • It was also developed to aid in the differentiation of lactose and non-lactose-fermenters from clinical specimens, suspected foods, and other such samples.

Limitations of SS Agar

  • The inclusion of green brilliant in this media is highly selective and has been proven to slow the growth of certain Shigella.
  • The bile salts could become crystallized in time. They appear as tiny like spider-like puff balls in the medium, and don’t interfere with the efficiency of the medium.
  • Certain varieties of Shigella such as Shigella sonnei as well as S. dysenteriae serovar 1 are able to ferment lactose slow, and the colonies shift to fermenting lactose after cultivation for at least 2 days.
  • A few non-pathogenic organisms may grow on Salmonella Shigella agar.
  • It is suggested that immunological, biochemical, mass spectrometry or molecular tests be conducted on the colonies of pure culture to confirm the identity of the colony.


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