Culture Media Preparation Tips

Salmonella Shigella agar preparation

Written by MicroDok

Salmonella-Shigella agar (SSA) is used for the selective cultivation and isolation of strains of Shigella species and Salmonella from clinically important samples. It is both a selective and differential media; and it can also be used to recover these organisms from environmental or non-clinical samples such as contaminated food samples. Another bacteriological media that performs similar function as the Salmonella-Shigella agar is Desoxycholate Citrate Agar (DCA). DCA can also be used for the selective isolation of Salmonella and Shigella species from both clinical and non-clinical samples. SSA or DCA is inhibitory in nature; and these bacteriological media contain some chemicals such as bile salt, sodium citrate and brilliant green that inhibit the growth of some commensal organisms or normal flora in the gastrointestinal tract, including Proteus and Gram positive bacteria that may be contaminants in the sample.

This image shows how Salmonella-Shigella agar looks like after preparation

Components of Salmonella-Shigella agar base

The components of Salmonella-Shigella agar base required for Salmonella-Shigella agar preparation include:

  1. Agar – which is the solidifying agent
  2. Beef extract – which provide sources of nitrogen, carbon, and vitamins
  3. pH – which is usually adjusted to 7.0 at 25 °C (or 77 °F)
  4. Lactose – which is the source of carbohydrate or carbon
  5. Bile salts – which serve to inhibit the growth of Gram-positive bacteria, Proteus, coliform organisms
  6. Sodium Citrate – which serve to inhibit the growth of Gram-positive bacteria, Proteus, coliform organisms
  7. Neutral red – which turns red in the presence of an acidic pH. This shows that fermentation of lactose has occurred.
  8. Ferric Citrate – which allows for the detection of hydrogen sulfide by the production of colonies with black colouration
  9. Enzymatic digest of animal tissue – which provide sources of nitrogen, carbon, and vitamins
  10. Enzymatic digest of casein – which provide sources of nitrogen, carbon, and vitamins
  11. Sodium thiosulphate – which allows for the detection of hydrogen sulfide by the production of colonies with black colouration
  12. Brilliant green – which serve to inhibit the growth of Gram-positive bacteria, Proteus, coliform organisms

MATERIALS

You require these materials to prepare your Salmonella-Shigella agar: Salmonella-Shigella agar powder/base (usually comes in 500 g), autoclave, conical flask, measuring cylinder, beaker, stirring rod, Bunsen burner, incubator, refrigerator, wire gauze, spatula, weighing balance, timer, cotton wool, aluminium foil, distilled water, Petri dish

STEP BY STEP PROTOCOL TO PREPARE SALMONELLA-SHIGELLA AGAR

  1. Weigh out 60.00 g of Salmonella-Shigella agar powder using the weighing balance.
  2. Suspend the 60.00 g of Salmonella-Shigella agar (SSA) powder in 1 litre (1000 ml) of distilled water.
  3. Mix the solution by stirring to dissolve the agar.
  4. Heat the mixture by boiling to dissolve the SSA powder completely. Monitor the boiling process closely in order to avoid charring the agar.
  5. Do not autoclave SSA medium. SSA is not autoclaved because overheating destroys the efficacy of the chemicals (e.g. bile salts and sodium citrate) that improves the selectivity of the media.
  6. Allow molten SSA medium to cool to about 45-50 degrees Celsius after boiling.
  7. Pour prepared molten medium into sterile Petri dish plates.
  8. Allow the poured plates on the bench to solidify.
  9. Do sterility check by incubating the poured plates in the incubator at 37 degrees Celsius for 18-24 h.
  10. At the end of incubation, check the plates for any sign of microbial growth (which is usually indicated by the presence of colony).
  11. Absence of colony on the plate means that your sterilization is good.
  12. You can now use your prepared Salmonella-Shigella agar plates for your experiment OR store in the refrigerator at 4 degrees Celsius until use.

REFERENCES

  1. Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. Part 2 . Cambridge University Press, UK.

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MicroDok

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