Chocolate agar is a chocolate-brown colored medium as a result of red blood cell lysis. It is used primarily to isolate fastidious organisms like H. influenza.

example of a sterile chocolate agar image

Image 1: The image above is an example of a sterile chocolate agar.
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Difference between blood agar and chocolate agar

There is really nothing much of a difference between the two. In fact, the compositions are the same. The manner of preparation is the difference between them as chocolate agar requires lysis of the red blood cells when added to the molten agar base.

As a result of RBC lysis, intracellular nutrients are released such as hemoglobin, coenzyme nicotinamide adenine dinucleotide, and hemin into the agar which is used by fastidious organisms like H. influenza. It is the lysis of the red blood cells that give the medium its chocolate-brown color; hence, the reason for the name chocolate agar. (1, 2, 3, and 4)

comparison image between chocolate and blood agar

Image 2: A comparison image between chocolate and blood agar.
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Composition of chocolate agar?

  • Proteose peptone
  • Dextrose
  • Sodium chloride
  • Disodium phosphate
  • Agar
  • Final pH

How to prepare a chocolate agar?

  • A volume of blood either from a horse or sheep is heat-lyse very slowly in a water bath at a 56 degrees Celsius. The blood must only be 5% of the total media volume.
  • Place about 20 ml into the Petri dish and let it solidify and condensation to dry.
  • Put the plate in a sterile plastic and store it at a temperature of 4 degrees Celsius until use. Before using the medium, it should be warmed to room temperature at a 25 degrees Celsius.
  • To test for sterility, the uninoculated plate should be incubated for 48 hours at a temperature of 35 degrees Celsius. (2, 4, and 5)

Medium preparation

  • Add all the components except for the hemoglobin solution to the distilled water. The total volume should be 500 ml and should be thoroughly mixed. Bring to boil and autoclave for about 15 minutes at 12 pounds pressure at a 21 degrees Celsius. Allow to cool at 45 degrees Celsius. About 500 mL of the hemoglobin solution should be added. Gently mix and pour into the sterile Petri dish. (2, 6, and 7)

Principle of chocolate agar

If a supplement is added on a chocolate agar base, it would be a perfect growth place for fastidious organisms. For the organism to grow, various nutrients are added such as casein and animal tissue, which serves as amino acids, nitrogenous nutrients, and other vital elements for the organism’s growth. (1, 4, 5, and 7)

Modifications can be made to grow other organisms. Some of the modifications include:

  • Thayer-Martin media – It is a chocolate agar supplemented with nystatin, vancomycin, and colistin. The purpose is to inhibit the normal flora and non-pathogenic Neisseria for the isolation of N. meningitides and N. gonorrhoeae.
  • Chocolate agar with bacitracin – It is a selective medium for the improvement of primary isolation of H. influenzae from a specimen that has a mixed flora of bacteria and fungi.
  • Chocolate agar with GC base and growth supplement – It supports the growth requirements needed to isolate fastidious organisms. (1, 3, 8, and 9)

What is the purpose of chocolate agar?

Obvious colonies of bacteria on chocolate agar

Chocolate agar is used to isolate and cultivate fastidious microorganisms such as Neisseria species and Haemophilus species. A chocolate agar is also useful in isolating N. gonorrheae from both acute and chronic cases of gonococcal infections. (2, 5, and 7)

Image 3: Obvious colonies of bacteria on chocolate agar.
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Growth of Neisseria on chocolate agar

Image 4: Growth of Neisseria on chocolate agar.
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Colony characteristics in chocolate agar

Neisseria meningitides

Observe the chocolate agar medium for the following colony characteristics:

  • Round, large, smooth, convex colorless to grey colony.
  • The colonies appear opaque on the plate with no obvious medium discoloration.
  • The colonies glistened with a defined edge. (3, 6, 9, and 10)

S. pneumonia

The chocolate agar plate has small greyish to greenish colonies with the alpha-hemolysis zone.

growth of N. gonorrhoeae on chocolate agar

Image 5: A growth of N. gonorrhoeae on chocolate agar.
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Neisseria gonorrhoeae

You will observe pinkish to brownish translucent colonies. They have a smooth consistency with a defined margin.

H. influenza colonies on a chocolate agar

Image 6: H. influenza colonies on a chocolate agar.
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Haemophilus influenza

The colonies appear to be non-hemolytic with opaque cream to grey in color.

Are there any limitations?

  1. For complete identification of the organism, an additional biochemical or serological test should be performed.
  2. Chocolate agar is an example of an enriched medium. There is a tendency that the pathogenic bacteria may be overgrown by the non-pathogenic ones. When isolating N. gonorrhoeae, a selective medium like Thayer Martin agar should be used.
  3. Whether N. gonorrhoeae is present or absent, it does not rule out the presence of other disease-causing microorganisms.
  4. Precipitated hemoglobin may look as dark spots on the media but it does not have any impact on the media’s performance. (2, 4, 9, and 10)

Quality control

If you are going to use a commercially prepared cultured media be it chocolate agar or other types of media, make sure you perform quality control testing. It is in accordance with the government regulatory agencies. You should check for signs and indicators of contamination and deterioration. A quality control testing should be done to check for growth or positive reactions and to demonstrate any negative reactions or inhibition. (2, 4, and 5)

What to keep in mind?

Chocolate agar is an enriched non-selective media and it needs supplementation to facilitate the growth of fastidious anaerobes. It needs to be used correctly to facilitate the growth of most pathogens in clinical infections. However, chocolate agar does not provide complete information for identifying bacterial isolates.

Additional tests have to be performed and another media is needed for complete identification. There are anaerobic microorganisms that are aero-tolerant and they usually grow well in anaerobic conditions but not so in air or carbon dioxide. (1, 6, and 7)



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