Preparation of MacConkey agar

MacConkey agar is both a selective and differential media.  By being a selective media, MacConkey agar can be used to selectively isolate a particular organism from a sample containing a mixture of microbes/bacteria. By being a differential media, MacConkey agar can be used to differentiate between two closely related bacteria. It is used for the isolation and differentiation of members of the Enterobacteriaceae family including E. coli, Salmonella, Shigella and Klebsiella that produces different morphological features on MacConkey agar. With MacConkey agar, we can differentiate lactose fermenters (e.g. E. coli) from non-lactose fermenters (e.g. Shigella and Salmonella).  Lactose fermenters produces a red/pink colony on MacConkey agar while non-lactose fermenters produces a colourless colony on MacConkey agar.

MacConkey agar contain some chemicals such as crystal violet and bile salts – which are both responsible for the selective nature of this bacteriological media. Crystal violet and bile salt are inhibitory to the growth of Gram positive bacteria such as S. aureus; and this defines the selective nature of MacConkey agar. However, MacConkey agar also contain another chemical known as neutral redNeutral red is a pH indicator that turns red at a pH below 6.8 and is colorless at any pH greater than 6.8. The presence of neutral red in MacConkey agar defines the differential nature of the media, since it can be used to differentiate between two closely related organisms as seen in members of the Enterobacteriaceae family as aforesaid.

This image shows how MacConkey agar looks like after preparation

Components of MacConkey agar base

The components of MacConkey agar base required for MacConkey agar preparation include:

  1. Agar – which is the solidifying agent
  2. Peptone – which provides nitrogen source
  3. Beef extract – which provides carbohydrates, vitamins
  4. Sodium chloride – which provides a physiological state akin to that of the living system
  5. pH – which is usually adjusted to 7.1 at 25 °C (or 77 °F)
  6. Lactose monohydrate – which is the source of carbohydrate or carbon
  7. Bile salts – which is the inhibitor of Gram positive bacteria
  8. Neutral red – which helps to differentiate lactose fermenting colonies from non-lactose fermenting colonies
  9. Crystal violet – which is also an inhibitor of Gram positive bacteria

MATERIALS

You require these materials to prepare your MacConkey agar : MacConkey 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 MacConkey Agar

  1. Weigh out 49.53 g of MacConkey agar powder using the weighing balance.
  2. Suspend the 49.53 g of MacConkey agar powder in 1 litre (1000 ml) of distilled water in a conical flask.
  3. Mix the solution by stirring to dissolve the agar.
  4. Bring the mixture to boil, by mild boiling of the mixture over a Bunsen burner flame. This helps to dissolve the agar completely. Monitor the boiling process closely in order to avoid charring the agar.
  5. Transfer the conical flask containing the boiled/mixed nutrient agar suspension to the autoclave.
  6. Sterilize the medium at 121 degrees Celsius at 15 psi (or 15 lbs of pressure) for 15 min in the autoclave.
  7. At the end of sterilization, allow the autoclave to return to normal (zero point) before opening. Otherwise the pressure built up in the autoclave will affect the quality and quantity of the prepared molten media. More so, you may be affected by the steam from the autoclave due to the high pressure built up in the autoclave.
  8. Allow molten medium to cool to about 45-50 degrees Celsius.
  9. Pour prepared molten medium into sterile Petri dish plates.
  10. Allow the poured plates on the bench to solidify.
  11. Do sterility check by incubating the poured plates in the incubator at 37 degrees Celsius for 18-24 h.
  12. At the end of incubation, check the plates for any sign of microbial growth (which is usually indicated by the presence of colony).
  13. Absence of colony on the plate means that your sterilization is good.
  14. You can now use your prepared MacConkey 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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