Chocolate Agar (also known as CHOC or CAP) is an enriched, non-selective bacterial growth medium used primarily for the isolation of fastidious organisms, particularly Haemophilus and Neisseria species. Despite the name, this medium contains no chocolate. Instead, it derives its name from the chocolate-brown color that results when red blood cells (RBCs) are lysed by heating. This lysis releases intracellular nutrients essential for the growth of demanding bacterial pathogens.
Principle of Chocolate Agar
The enhanced nutrient composition of chocolate agar supports the growth of fastidious bacteria. Here’s how it works:
- Casein and animal tissue digest: Provide nitrogen, amino acids, and essential growth elements.
- Corn starch: Neutralizes toxic fatty acids that can inhibit sensitive organisms like Neisseria.
- Potassium phosphates: Maintain pH stability during bacterial growth.
- Haemoglobin solution: Supplies the X factor (hemin) for Haemophilus species.
- IsovitaleX (coenzyme enrichment): Provides the V factor (nicotinamide adenine dinucleotide, NAD) and other essential vitamins for Neisseria growth.
Chocolate Agar Composition
Although similar to blood agar, chocolate agar is prepared by heating the blood component, which lyses the RBCs.
Below is the typical composition of commercially prepared chocolate agar:
| Ingredients | Quantity (g/L) |
| Casein/Animal tissue digest | 15 |
| Corn starch | 1 |
| Sodium chloride | 5 |
| Dipotassium phosphate | 4 |
| Monopotassium phosphate | 1 |
| Haemoglobin solution | 2% |
|
Coenzyme enrichment (IsovitaleX) |
10 mL |
| Agar | 10 |
Preparation of Chocolate Agar
Follow these steps for successful preparation:
- Prepare the base: Use a blood agar base according to the manufacturer’s instructions.
- Sterilize: Autoclave at 121°C for 15 minutes.
- Add blood: Mix 57% v/v of defibrinated sheep or horse blood.
- Heat: Incubate in a water bath at 75–80°C until the medium turns chocolate brown.
- Cool: Let the medium cool to 50–55°C before pouring into sterile Petri dishes.
- Label and store: Mark plates with the date and type; store inverted at 2–8°C until ready for use.
Colony Morphology of Microorganisms on Chocolate Agar
| Microorganism | Colony Morphology |
| Haemophilus influenzae | Small, moist, clear to grey, round colonies |
| Neisseria gonorrhoeae | Small, convex, glistening, greyish-white colonies |
| Neisseria meningitidis | Large, convex, grey colonies |
| Moraxella catarrhalis | Opaque, slightly elevated grey-white colonies |
| Brucella species | Tiny, translucent to white colonies after long incubation |
Uses of Chocolate Agar
It has diverse clinical and microbiological applications:
- Isolating fastidious respiratory pathogens, including Haemophilus influenzae.
- Culturing Neisseria species such as N. gonorrhoeae and N. meningitidis.
- Processing clinical samples like cerebrospinal fluid (CSF), throat swabs, genital swabs, and sputum.
- Enriching slow-growing microorganisms that don’t thrive on standard media.
- Diagnostic tool for diseases such as meningitis, epiglottitis, and gonorrhea.
Precautions When Using Chocolate Agar
- Avoid overheating: Excessive heat destroys critical growth factors (NAD and hemin).
- CO₂ requirement: Incubate in a 5–10% CO₂ environment for optimal growth.
- Prevent contamination: Work in sterile conditions and avoid drying the plates.
- Proper storage: Keep at 2–8°C and bring to room temperature before use
Limitations of Chocolate Agar
While effective, chocolate agar has a few limitations:
- Cannot differentiate lactose fermenters like MacConkey agar does.
- Not ideal for mixed cultures—overgrowth can mask target pathogens.
- Selective agents may be needed (e.g., Thayer-Martin agar for Neisseria).
- Costly and less stable due to the blood components compared to simpler media.
Conclusion
Chocolate agar is a critical tool in clinical microbiology, enabling the detection and isolation of fastidious organisms that do not grow on standard media. With proper preparation, handling, and use, it plays a vital role in diagnosing infections caused by Haemophilus, Neisseria, and other demanding pathogens.
