Chocolate Agar Media – Principle, Composition, Preparation, Result

What is Chocolate Agar Media?

• Chocolate agar media, also known as CAP or CHOC, is a specialized type of agar used in microbiology for the cultivation and identification of fastidious bacterial pathogens. It is very similar to blood agar, with one key difference: the red blood cells present in the medium are lysed during the preparation process. This lysis releases valuable intracellular nutrients such as hemoglobin, hemin (also known as the “X” factor), and the coenzyme nicotinamide adenine dinucleotide (NAD or the “V” factor) into the agar.
• The lysis of red blood cells in chocolate agar results in the medium acquiring a distinctive chocolate-brown color, from which it derives its name. This chocolate-brown coloration is an indicator that the medium is enriched with the necessary nutrients for the growth and survival of fastidious bacteria.
• Fastidious bacteria are microorganisms that have complex nutritional requirements and are more demanding in terms of their growth conditions compared to other bacteria. Examples of bacterial pathogens that commonly require the enriched environment provided by chocolate agar include Neisseria gonorrhoeae, the causative agent of gonorrhea, and various species of the Haemophilus genus.
• To prepare chocolate agar, blood agar is first heated until it becomes molten. This process causes the red blood cells within the agar to rupture, releasing their intracellular nutrients. The resulting medium provides the fastidious bacteria with the essential components they need for growth. This enriched medium is then poured into Petri dishes or other containers and allowed to solidify.
• In the laboratory, chocolate agar is frequently used to isolate and cultivate fastidious pathogens from clinical samples. The medium’s composition makes it highly supportive of the growth of bacteria that might not thrive on other types of media. By providing the necessary nutrients, chocolate agar enables the identification and further study of these bacteria, aiding in the diagnosis and treatment of associated infections.
• In summary, chocolate agar is a specialized agar medium used for the cultivation and isolation of fastidious bacteria. It is similar to blood agar but contains lysed red blood cells, releasing important nutrients for the growth of specific bacterial pathogens. The medium’s chocolate-brown coloration is a visual indicator of its enrichment, and it is particularly useful in supporting the growth of bacteria such as Neisseria gonorrhoeae and Haemophilus species.

Principle of Chocolate Agar

The principle of chocolate agar lies in its composition and supplements, which create an optimal growth environment for fastidious organisms while inhibiting the growth of contaminating organisms. The base of chocolate agar contains casein and animal tissue digest, which provide nitrogenous nutrients, amino acids, and other essential elements necessary for the growth of these organisms.

Neisseria species, in particular, are sensitive to toxic substances such as fatty acids. To counteract this, cornstarch is added to neutralize potential toxic metabolites, while potassium phosphate helps maintain a uniform pH during growth. Sodium chloride is included to maintain osmotic equilibrium, ensuring the integrity of cells.

Chocolate agar is essentially a variant of the blood agar plate. Red blood cells within the agar are lysed by slowly heating the medium to 80°C. This heat treatment inactivates enzymes that could degrade the essential coenzyme nicotinamide adenine dinucleotide (NAD). The lysed red blood cells provide the necessary X factor derived from hemoglobin and the V factor derived from a growth supplement, both of which are required by fastidious organisms for growth.

Modifications can be made to chocolate agar to support the growth of specific organisms. Thayer-Martin Media, for example, is a chocolate agar supplemented with vancomycin, nystatin, and colistin. These additives inhibit the growth of normal flora, including nonpathogenic Neisseria, making it a selective medium for the isolation of pathogenic Neisseria species such as N. gonorrhoeae and N. meningitidis.

Another modification involves supplementing chocolate agar with bacitracin. This selective medium is used to improve the primary isolation of Haem

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.

Ingredients	Gm/L
Casein/Animal Tissue Digest	15.0
Cornstarch	1.0
Potassium Phosphate, Dibasic	4.0
Potassium Phosphate, Monobasic	1.0
Sodium Chloride	5.0
Agar	10.0
Hemoglobin Solution (2%)	500.0 ml
Isovitox Enrichment	10.0 ml

Preparation of Chocolate Agar

The preparation of chocolate agar involves several steps to ensure the proper incorporation of hemoglobin and the growth-supporting components. Here is a detailed description of the preparation process:

1. Preparation of the hemoglobin solution:
   • Take hemoglobin and add it to distilled water.
   • Bring the volume to 500 ml and mix thoroughly.
   • Autoclave the solution for 15 minutes at 15 pounds of pressure at 121°C.
   • Allow the solution to cool to 45-50°C.
2. Preparation of the medium:
   • Add all the components of the chocolate agar, except for the hemoglobin solution, to distilled water.
   • Bring the volume to 500 ml and mix thoroughly.
   • Gently heat the mixture until it starts boiling.
   • Autoclave the medium for 15 minutes at 15 pounds of pressure at 121°C.
   • Allow the medium to cool to 45-50°C.
   • Add 500 ml of the sterile hemoglobin solution to the medium and mix thoroughly.
3. Pouring the agar:
   • Prepare the blood agar base according to the manufacturer’s instructions.
   • Autoclave the blood agar base at 121°C for 15 minutes.
   • Add 5-7% v/v of defibrinated blood (horse or sheep blood) to the sterilized blood agar base.
   • Place the mixture in a water bath at 75-80°C and gently swirl until the color changes to dark brown.
   • Once the mixture has cooled to 50-55°C, pour it into sterile Petri plates under aseptic conditions.
   • Label the plates with the name of the media and the date of preparation.
   • Store the plates inverted at 2-8°C until they are ready to be used.
4. Preparation of chocolate agar slants:
   • Dispense 4 ml of the prepared chocolate agar medium into 16×125 mm screw-cap tubes.
   • Keep the tubes in a slanted position to allow the medium to solidify.
   • The chocolate agar slants will have a brown to brownish-red color.
   • Store the slants at 4°C when not in use, and warm them to room temperature (25°C) before use.

Following these steps will result in properly prepared chocolate agar plates and slants that provide an enriched growth medium for the cultivation of fastidious organisms in the laboratory.

Result interpretation and Colony Morphology

The interpretation of results on chocolate agar involves examining the characteristics of colonies formed by specific organisms. Here is a description of the colony characteristics of some common bacteria on chocolate agar:

1. Neisseria gonorrhoeae:
   • Colonies appear small, grey to white in color.
   • They have a mucoid texture with a smooth consistency.
   • The margins of the colonies are well-defined.
2. Neisseria meningitidis:
   • Colonies are larger in size compared to Neisseria gonorrhoeae.
   • They appear bluish-grey in color.
   • The colonies have a mucoid texture and are round and convex in shape.
   • The surface of the colonies is smooth, moist, and glistening.
   • The edges of the colonies are clearly defined.
3. Haemophilus influenzae:
   • Colonies formed by Haemophilus influenzae are small in size.
   • They appear colorless, lacking pigmentation.
   • The colonies have a moist texture.
   • Haemophilus influenzae colonies often have a characteristic seminal odor.

Interpreting these colony characteristics on chocolate agar can help in the identification of specific bacteria. Neisseria gonorrhoeae colonies, for example, have distinct features that differentiate them from Neisseria meningitidis colonies. Similarly, the colony characteristics of Haemophilus influenzae on chocolate agar can aid in its identification.

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

Quality control of media

Quality control of media is an essential step in ensuring the reliability and consistency of the prepared agar. Here is a description of the quality control procedures for chocolate agar:

1. Sterility testing:
   • Incubate 3-5 uninoculated plates from each batch of prepared media.
   • The plates should be incubated at 37°C for 18-24 hours.
   • Any growth observed on the plates indicates a positive result, indicating contamination.
   • If contamination is detected, the entire batch of media should be discarded.
2. Performance testing:
   • Inoculate prepared plates with specific organisms to assess the growth performance of the completed medium.
   • For chocolate agar, the recommended organisms for performance testing are Neisseria gonorrhoeae ATCC 43069 and Haemophilus influenzae ATCC 10211.
   • Inoculate each organism onto separate plates.
   • Incubate the plates at 35-37°C with 5% CO2 (or in a candle jar providing up to 3% CO2) for 18-24 hours.
   • Observe and compare the growth characteristics of the organisms with the expected results.

Expected results for performance testing on chocolate agar:

• Neisseria gonorrhoeae ATCC 43069: Luxuriant growth with small, grey to white colonies.
• Haemophilus influenzae ATCC 10211: Good growth with small, colorless, moist colonies.

By performing sterility testing and assessing the growth performance of specific organisms, laboratories can ensure that the prepared chocolate agar meets the required quality standards. These quality control measures help identify any potential contamination or issues with the growth characteristics of the medium, ensuring accurate and reliable results in microbiological testing.

Organism Tested	Results in 5% CO2	Results in anaerobic conditions	Time
Fusobacterium necrophorum	No Growth	Growth	24 – 48 hrs.
Neisseria gonorrhoeae*	Growth	Inhibited Growth	48 hrs.
Haemophilus influenzae*	Growth	Growth	24 – 48 hrs.
Streptococcus pneumoniae	Growth	Growth	24 – 48 hrs.
Staphylococcus aureus	Growth	Growth	24 – 48 hrs.
Escherichia coli	Growth	Growth	24 hrs.

Uses of chocolate agar

Chocolate agar has various uses in microbiology, particularly in the isolation and cultivation of fastidious microorganisms. Here are some key applications of chocolate agar:

1. Isolation of fastidious organisms: Chocolate agar is commonly used to isolate and cultivate fastidious organisms such as Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis from various clinical specimens. These organisms have specific growth requirements that are met by the enriched medium provided by chocolate agar. Isolation of these organisms aids in the diagnosis of diseases caused by them.
2. Screening for Haemophilus influenzae: Chocolate agar supplemented with bacitracin can act as a selective medium for screening Haemophilus influenzae from specimens that contain a mixed flora of microorganisms, such as sputum samples. Bacitracin inhibits the growth of certain bacteria, allowing for the isolation and identification of Haemophilus influenzae.
3. Cultivation of Haemophilus and Neisseria species: Chocolate agar provides an enriched growth medium suitable for the cultivation of Haemophilus and Neisseria species. These organisms often have complex nutritional requirements and may require specific growth factors, which are provided by the lysed red blood cells in chocolate agar. It enables the isolation, identification, and further study of these bacteria.
4. Isolation of Neisseria gonorrhoeae: Chocolate agar is commonly used for the isolation of Neisseria gonorrhoeae from both chronic and acute cases of gonococcal infections. The enriched medium supports the growth of Neisseria gonorrhoeae, allowing for its isolation and subsequent characterization.

In summary, chocolate agar finds wide applications in the isolation and cultivation of fastidious microorganisms, including Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis. Its enriched composition provides the necessary nutrients and growth factors required by these organisms, aiding in the diagnosis and study of associated infections. Additionally, chocolate agar with bacitracin can act as a selective medium for screening Haemophilus influenzae from specimens containing mixed microbial flora.

Limitations of Chocolate Agar

While chocolate agar is a valuable medium for the isolation and cultivation of fastidious organisms, it does have certain limitations. Here are some limitations associated with the use of chocolate agar:

1. Additional tests for identification: While chocolate agar provides a favorable environment for the growth of fastidious organisms, it is important to note that further biochemical and/or serological tests are usually required to complete the identification of the isolated colonies. This is because the characteristics observed on the chocolate agar alone may not be sufficient for definitive identification.
2. Overgrowth of non-pathogenic organisms: Since chocolate agar is an enriched medium, non-pathogenic organisms may overgrow pathogenic bacteria. This can make it challenging to differentiate between non-pathogenic organisms and pathogenic ones solely based on their growth characteristics on chocolate agar. For specific isolation of Neisseria gonorrhoeae, a selective medium like Thayer Martin Agar, Modified is recommended in parallel with chocolate agar.
3. Presence of precipitated hemoglobin: Precipitated hemoglobin may appear as dark spots on or in the chocolate agar, but this does not impact the performance of the medium. These spots are harmless and do not interfere with the growth or identification of the target organisms.
4. Limitations in ruling out other pathogens: The presence or absence of Neisseria gonorrhoeae in a specimen does not rule out the possibility of other pathogenic organisms being present. Chocolate agar specifically supports the growth of fastidious bacteria, and additional culture methods or tests may be required to identify other potential pathogens.
5. Susceptibility to contamination: Chocolate agar, being an enriched medium, is prone to contamination. Careful aseptic techniques should be followed during the preparation and handling of chocolate agar plates to minimize the risk of contamination.
6. Fragility of the plate surface: Chocolate agar has a reduced concentration of agar, making the surface of the plate fragile and more susceptible to scratches during streaking or manipulation. This can impact the isolation and growth of desired organisms and may necessitate additional precautions while handling the plates.

Despite these limitations, chocolate agar remains a valuable tool in the laboratory for the isolation and cultivation of fastidious organisms, with careful interpretation and complementation with other tests to ensure accurate identification and diagnosis.

Modifications of Chocolate Agar

Modifications of chocolate agar have been developed to enhance its selectivity and support the growth requirements of specific organisms. Here are some notable modifications:

1. Thayer-Martin Media: Thayer-Martin medium is a modified version of chocolate agar supplemented with vancomycin, nystatin, and colistin. These additives inhibit the growth of normal flora, including nonpathogenic Neisseria species. Thayer-Martin medium is specifically designed for the selective isolation of pathogenic Neisseria gonorrhoeae and Neisseria meningitidis.
2. Chocolate Agar with bacitracin: This modification of chocolate agar includes the addition of bacitracin. Bacitracin acts as a selective agent, improving the primary isolation of Haemophilus influenzae from specimens that contain a mixed flora of bacteria and/or fungi, such as sputum samples.
3. Chocolate agar supplemented with GC base and growth supplement: This variant of chocolate agar is specifically formulated to support the growth requirements of Haemophilus species. It includes GC base (containing hemin) and a growth supplement (containing NAD). This modification provides the necessary factors for the isolation of Haemophilus species when incubated at 35-37°C in a 5% CO2 atmosphere.
4. Chocolate agar with TSA and growth supplements: This modification of chocolate agar includes the addition of tryptic soy agar (TSA) and growth supplements. The growth supplements typically contain hemin and NAD, supporting the growth requirements of fastidious organisms, such as Haemophilus influenzae. This modification is particularly useful for the isolation of these organisms when incubated at 35-37°C in a 5% CO2 atmosphere.

These modifications of chocolate agar offer increased selectivity and provide the specific growth factors required by certain bacteria. By tailoring the composition of the medium, these modifications allow for improved isolation and cultivation of target organisms in the laboratory, aiding in the diagnosis and study of associated infections.

FAQ

References

1. https://microbeonline.com/chocolate-agar-composition-uses-colony-characteristics
2. https://en.wikipedia.org/wiki/Chocolate_agar
3. https://assets.fishersci.com/TFS-Assets/LSG/manuals/IFU1300.pdf
4. https://www.dalynn.com/dyn/ck_assets/files/tech/PC55.pdf
5. https://anaerobesystems.com/products/plated-media/chocolate-agar-choc/
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC179834/
7. https://aimed.net.au/2016/03/27/chocolate-agar-for-easter/
8. https://hardydiagnostics.com/media/assets/product/documents/ChocolateAgar.pdf