Chocolate agar preparation

Chocolate agar (CA) is used for the selective cultivation and isolation of fastidious organisms. The organisms that can be isolated with chocolate agar include Streptococcus pyogenes, Haemophilus, Francisella and Neisseria. Chocolate agar is prepared from molten blood agar base after adding 5 % defribinated sheep blood; and the protocol for preparing chocolate agar is similar to the steps involved in preparing blood agar. Only a minor modification exist in the protocol. Chocolate agar like blood agar is a non-selective but enriched or enrichment growth media used for the selective isolation of fastidious bacteria as aforesaid. The presence of blood in chocolate agar is what makes it to be enriched in nature. Chocolate agar can also be called boiled blood agar, because chocolate agar is prepared from blood agar by a mild heating of molten blood agar after adding blood to the molten blood agar base.

                This image shows how Chocolate agar looks like after preparation

Components of blood agar base

NOTE: Chocolate agar is prepared from molten blood agar, with only a slight modification in the protocol for preparing blood agar. Therefore, the protocol for preparing blood agar is elaborated in this section, while noting the slight modification that converts molten blood agar into chocolate agar (in number 12 of the preparation protocol below). The components of blood agar base required for blood agar preparation include:

  1. Blood agar base- which is the solidifying agent
  2. Beef/meat extract – which provide sources of nitrogen, carbon, and vitamins
  3. pH – which is usually adjusted to 7.3 at 25 °C (or 77 °F)
  4. Peptone – which serves as source of vitamins
  5. Sodium chloride – for maintaining a balanced physiological environment as obtainable in living systems

You require these materials to prepare your blood agar: blood agar base or nutrient agar base (usually comes in 500 g), 5-10 % of sterile defibrinated sheep blood, 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 CHOCOLATE AGAR

  1. Weigh out 40.00 g of blood agar powder using the weighing balance.
  2. Suspend the 40.00 g of blood 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 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 medium. 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 blood agar medium to cool to about 45-50 degrees Celsius.
  9. Add about 5-10 % of sterile defibrinated sheep blood.
  10. Stir /mix the molten blood agar vigorously.
  11. Heat the molten blood agar over a Bunsen burner flame mildly. Do this until the blood agar turns brown. [This is the step that differentiates blood agar preparation from chocolate agar preparation]
  12. Pour prepared molten chocolate agar medium into sterile Petri dish plates.
  13. Allow the poured plates on the bench to solidify.
  14. Do sterility check by incubating the poured plates in the incubator at 37 degrees Celsius for 18-24 h.
  15. At the end of incubation, check the plates for any sign of microbial growth (which is usually indicated by the presence of colony).
  16. Absence of colony on the plate means that your sterilization is good.
  17. You can now use your prepared Chocolate 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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