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Chocolate Agar Media Principle, Composition, Preparation, Result

Chocolate Agar (CAP or CHOC) is a nonselective enhanced medium that is used to identify and isolate of pathogens that are fastidious.

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MN Editors

Chocolate Agar Media

  • Chocolate Agar (CAP or CHOC) is a nonselective enhanced medium that is used to identify and isolate of pathogens that are fastidious.
  • Chocolate agar is made through heating the blood agar which then breaks the red blood cell (RBC) which releases its nutrients to aid in the development of fastidious bacteria particularly Haemophilus as well as Neisseria species.
  • The name derives because elimination of RBC results in the color of chocolate brown.
  • The chocolate agar is basically identical to blood agar, except that during the preparation process, the red blood cells get killed when they are added to the molten agar base. This results in Cell lysis release intracellular nutrition like hemoglobin, Hemin (“X” factors) as well as the coenzyme nicotinamide-adenine dinucleot (NAD and “V” factor) into the agar, which is then utilized by fastidious bacteria.

Principle of Chocolate Agar

Chocolate Agar Base, with the addition of supplements will allow for a rapid growth of fastidious species without overgrowth due to contamination of organisms. The digest of animal tissues and Casein provide the organism with amino acids, nitrogenous nutrients as well as other essential elements to the growth of organisms. Because Neisseria species are extremely sensitive to harmful substances like fat acids, the addition of cornstarch is a way to neutralize toxic metabolites while potassium phosphate assists in maintaining an even pH throughout development.

The presence of sodium chloride can maintain the equilibrium of osmotic pressure and thus maintains cell integrity. Chocolate agar is also regarded as a variant of blood agar plate. It contains the red blood cells which have been killed by heating slowly to 80degC. The heat activates enzymes that might otherwise destroy NAD. The supplements added to the diet provide the factors X (from Haemoglobin) and V factor (from Growth Supplement) needed by extremely stricked organisms.

Certain modifications may also be made to enhance the growth of the particular organism. Thayer-Martin Media can be described as a chocolate Agar that is supplemented with vancomycin colistin and nystatin to block the normal flora and Neisseria that is nonpathogenic to aid in the specific removal of N. the gonorrhoeae, and N. meningitidis. Chocolate Agar that is supplemented with bacitracin can be a specific medium that is used to enhance the primary determination of H. influenzae from samples that contain a mix of fungi and/or bacteria. Chocolate agar supplemented by GC basis and growth supplements is yet another modification that assists in meeting the specific requirements for growth (hemin as well as NAD) necessary for the identification of fastidious organisms like H. influenzae, when it is it is incubated at 35 to 37 degrees Celsius in a environment of 5% CO2.

Composition of Chocolate Agar

The chemical composition that chocolate agar has is same as blood Agar. There is only one difference: that blood Agar gets heated up in an water bath to make chocolate agar. The commercially available chocolate agar is, however, of an entirely different composition and no blood addition.

Casein/Animal Tissue Digest15.0
Potassium Phosphate, Dibasic4.0
Potassium Phosphate, Monobasic1.0
Sodium Chloride5.0
Hemoglobin Solution (2%)500.0 ml
Isovitox Enrichment10.0 ml

Preparation of Chocolate Agar

  1. Make blood Agar. (See the steps to prepare blood Agar)
  2. Add 5-7% v/v the defibrinated part of your blood (horse or sheep blood) and then place the medium in a bath that ranges from 75 to 80 degrees and then continue swirling until the color shifts to dark brown.
  3. Pour the media into sterilized Petri plates in safe conditions once the media is cooled to 50 to 55 degrees Celsius.
  4. The plates should be labeled with the names of the medium, as well as the date of creation, and then place them on a rack at 2-8 degrees Celsius until they are needed.

Note: other supplements, such as isovitalex can be added to the preparation.

Chocolate agar slants

  1. Dispense 4 mL medium into 16X125 mm screw cap tubes
  2. Make sure the tubes remain in the an slanted direction and allow them to solidify
  3. Slants of chocolate appear to be in a brownish to reddish color.
  4. They should be kept at 4°C when not in use and then heated to ambient temperature (25degC) before using.

Result interpretation

Most organisms thrive on chocolate agar, forming grey colonies that vary in size. But, as it is specifically used to identify pathogens that are fastidious like Neisseria and Haemophilus and their colony morphology are described below.

OrganismColony morphology
Neisseria gonorrhoeaepinkish-brown and translucent, exhibit smooth consistency and defined margins, and are typically 0.5-1 mm in diameter
N. meningitidisgrayish, non-hemolytic, round, convex, smooth, moist, glistening colonies with a clearly defined edge, larger in size as compared to N.gonorrhoeae
Haemophilus influenzaeNon hemolytic, colorless, moist colonies with a characteristic “mousy” odour.

Quality control of media

Conduct sterility testing on the media prepared by incubating 3 uninoculated plates from each batch at 37degC over 18-24 hours. Any increase in the number of cells should be considered as positive and the entire batch should be removed.

The performance testing of the media should be conducted by inoculating these organisms on plates and incubating for 18-24 hours in a temperature range of 35 to 37 degrees using 5%CO2 (or in the candle jar, but it will only give 33 percent CO2) to assess the performance of the final medium.

OrganismExpected Result
Neisseria gonorrhoeae ATCC 43069Luxuriant growth, small, grey to white colonies
Haemophilus influenzae ATCC 10211Good  growth, small, colorless, moist colonies

Uses of chocolate agar

  • It is utilized for the isolation and cultivation of fastidious microorganisms, notably Haemophilus and Neisseria species.
  • It is used to aid in the identification of Neisseria gonococcus from acute and chronic instances of gonococcal infection.

Limitations of Chocolate Agar

  • It is suggested that further tests for serology or biochemistry be carried out on the isolated colonies from the culture that is pure in order to confirm the identity of the colony.
  • Chocolate Agar is an enriched media, so non-pathogenic organisms can grow faster than pathogenic bacteria. If the isolation of N. Gonorrhoeae is a goal an aerated medium that is selective like Thayer Martin Agar, Modified is recommended to be used in conjunction with this formula that is non-selective.
  • Hemoglobin that is precipitated may show up as dark spots in the media but is not a hindrance to the performance of the media.
  • The absence or presence of N. Gonorrhoeae on a specimen is not a way to disprove the existence of pathogenic organisms.

Modifications of Chocolate Agar

  • Thayer Martin Media Thayer martin is modified chocolate agar containing vancomycin, Nystatin, and colistin, to suppress the normal flora. This includes Neisseria that is nonpathogenic, allowing the specific elimination of N. Gonorrhoeae as well as N. meningitidis.
  • Chocolate Agar with Bacitracin: This modification serves for a particular medium that can enhance the primary separation of H. influenzae from samples like sputum which has an array of fungi and bacteria.
  • Chocolate agar supplemented by GC bases and growth supplements This type of agar can be used to help meet the specific requirements for growth (hemin as well as NAD) required to isolate Haemophilus spp. When incubated at 35-37°C in an atmosphere of 5% CO2.
  • Chocolate Agar with TSA along with growth and growth boosters: It’s an adaptation of chocolate agar which supports the specific needs for growth (hemin as well as NAD) required to isolate aggressive organisms like H. influenzae, when it is placed in a 35-37 degree C environment of 5% CO2.



MN Editors. (November 16, 2021).Chocolate Agar Media Principle, Composition, Preparation, Result. Retrieved from https://microbiologynote.com/chocolate-agar-media/


MN Editors. "Chocolate Agar Media Principle, Composition, Preparation, Result." Microbiology Note, Microbiologynote.com, November 16, 2021.


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