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Chocolate Agar

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.

Chocolate Agar
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Difference between blood agar and chocolate agar:

There really isn’t much difference between the two. In fact, the compositions are the same. The form of preparation is the difference between them, since chocolate agar requires lysis of red blood cells when added to the molten agar base.

As a result of the lysis of the red blood cells, intracellular nutrients such as hemoglobin, coenzyme nicotinamide adenine dinucleotide and hemin are released in the agar used by demanding organisms such as H. influenza. It is the lysis of the red blood cells that gives the medium its chocolate brown color; Therefore, the reason for the name chocolate agar.

Composition of chocolate agar?

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.

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.

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.

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. influenza 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.

Purpose of 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.

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.

S. pneumonia

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

Neisseria gonorrhoeae

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

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.

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