Overview:
Chocolate Agar Base (also known as Blood Agar Base for Chocolate Agar) is a highly nutritious, non-selective enriched solid medium designed for the isolation and cultivation of fastidious microorganisms, particularly Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis, which require hemin (X-factor) and NAD (V-factor) for growth. Developed as an extension of traditional blood agar, it is prepared by incorporating 10% defibrinated blood into the base and heating (80�C for 10 minutes) to lyse red blood cells, releasing essential growth factors and resulting in the characteristic chocolate-brown color. This medium supports the growth of a wide range of pathogens from clinical specimens like cerebrospinal fluid, blood, and genital swabs, while allowing observation of hemolytic reactions. It is a cornerstone in clinical microbiology per CLSI and ATCC protocols, often used without antibiotics for primary isolation.
Applications:
?? Isolation and cultivation of fastidious pathogens such as Haemophilus spp. and Neisseria spp. from clinical samples (e.g., CSF, throat swabs, genital secretions)
?? Primary recovery of Streptococcus pneumoniae, beta-hemolytic streptococci, and other respiratory tract pathogens
?? Support for antimicrobial susceptibility testing and vaccine production strains
?? General-purpose enrichment for mixed flora in blood cultures or environmental monitoring
Key Features:
?? Nutrient-rich formulation with meat extract and peptone provides amino acids, vitamins, and carbohydrates for optimal fastidious growth
?? Blood lysis releases X- and V-factors, enabling >90% recovery of ATCC strains like N. gonorrhoeae 49226 without supplementation
?? Non-selective but versatile; hemolytic zones (alpha, beta, gamma) visible for presumptive identification
?? Validated in USP/EP and ISO standards; low reducing sugars prevent interference with beta-hemolysis
?? Opaque, chocolate-brown appearance post-heating; stable for 35-37�C incubation under 5-10% CO?
Composition (per liter):
Meat Extract � 10.0 g
Peptone � 10.0 g
Sodium Chloride (NaCl) � 5.0 g
Agar � 15.0 g
Defibrinated Blood (10%, heated) � 100 mL
Final pH: 7.3 � 0.2 at 25�C
Preparation:
Suspend 40.0 g of the dehydrated base in 1 liter of purified/distilled water. Heat with frequent agitation to boiling until completely dissolved (do not overheat). Autoclave at 121�C (15 psi) for 15 minutes. Cool to 45-50�C in a water bath. Aseptically add 100 mL sterile defibrinated sheep or horse blood (10%). Mix thoroughly, then heat the mixture at 80�C for 10 minutes with constant gentle agitation to lyse erythrocytes and release growth factors (do not boil). Cool to 45-50�C and pour into sterile Petri plates (20-25 mL per plate). Allow to solidify at room temperature. The prepared medium should appear smooth, chocolate-brown, and opaque without bubbles or separation. Inoculate and incubate at 35-37�C in 5-10% CO? for 24-48 hours.
Storage:
Dehydrated medium: Store in a tightly closed container at 2�25�C, protected from moisture, light, and humidity; stable until expiry date (typically 3 years).
Prepared plates: Store at 2�8�C in sealed plastic bags; use within 2-4 weeks. Discard if dehydrated, hemolyzed, discolored, or contaminated.
?? For laboratory and research use only. Not for diagnostic procedures without proper validation. Not for human or veterinary consumption. Handle under biosafety level 2; use Universal Precautions due to animal blood�wear gloves, lab coat, and eye protection. Autoclave all waste before disposal. Blood source may affect hemolytic patterns; sheep blood preferred for streptococci. Suboptimal heating may reduce factor release and inhibit growth.