CHOCOLATE AGAR

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Chocolate agar (CA) is used for the selective cultivation and isolation of fastidious organisms (Figure 1). 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 exists in the protocol. Chocolate agar like blood agar is a non-selective but enriched growth media used for the selection 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.

Fugure 1. This image shows how Chocolate agar looks like after preparation. Photo courtesy: https://www.microbiologyclass.com

Components of Chocolate agar

The components of Chocolate agar base required for Chocolate 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 7.3 at 25 °C (or 77 °F)
  4. Lactose – source of carbohydrate or carbon
  5. Bile salts – serve to inhibit the growth of Gram-positive, Proteus, coliform organisms
  6. Sodium Citrate – serve to inhibit the growth of Gram-positive, 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 – serve to inhibit the growth of Gram-positive, Proteus, coliform organisms

MATERIALS

You require these materials to prepare your Chocolate agar: chocolate agar  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 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.
  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. 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.
  6. Transfer the conical flask containing the boiled/mixed nutrient agar suspension to the autoclave.
  7. Sterilize the medium at 121 degrees Celsius at 15 psi (or 15 lbs of pressure) for 15 min in the autoclave.
  8. 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 prepared molten medium which might pour out. More so, you can also be affected by the high pressure built up in the autoclave.
  9. Allow molten blood agar medium to cool to about 50 degrees Celsius.
  10. Pour prepared molten medium into sterile Petri dish plates.
  11. Allow the poured plates on the bench to solidify.
  12. Add about 5-10 % of sterile defibrinated sheep blood.
  13. Stir /mix the molten blood agar vigorously.
  14. Pour prepared molten medium into sterile Petri dish plates.
  15. Allow the poured plates on the bench to solidify.
  16. Do sterility check by incubating the poured plates in the incubator at 37 degrees Celsius for 18-24 h.
  17. At the end of incubation, check the plates for any sign of microbial growth (which is usually indicated by the presence of colony).
  18. Absence of colony on the plate means that your sterilization is good.
  19. You can now use your prepared Chocolate agar plates for your experiment OR store in the refrigerator at 4 degrees Celsius until use.

Further reading

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23rd edition. McGraw Hill Publishers. USA.

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

Goldman E and Green L.H (2008). Practical Handbook of Microbiology, Second Edition. CRC Press, Taylor and Francis Group, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.

Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.

Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

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