Overview:
Chocolate II Agar Base is an enriched, non-selective solid medium specifically formulated for the primary isolation and cultivation of fastidious microorganisms, particularly Neisseria species (e.g., N. gonorrhoeae, N. meningitidis) and Haemophilus species (e.g., H. influenzae, H. parainfluenzae), from clinical specimens such as urogenital swabs, cerebrospinal fluid, and respiratory samples. Developed by BD BBL as an improvement over standard chocolate agar, it utilizes a GC II Agar Base enriched with hemoglobin (providing X-factor/hemin) and IsoVitaleX Enrichment (supplying V-factor/NAD, vitamins, amino acids, coenzymes, and dextrose) to meet the exacting nutritional requirements of these pathogens. The medium neutralizes toxic metabolites via cornstarch and maintains a buffered pH for optimal growth under 3-5% CO? at 35-37�C, yielding small, grayish-white to mucoid colonies after 18-48 hours. It complies with CLSI and CDC guidelines for antimicrobial susceptibility testing and primary diagnostics.
Applications:
?? Primary isolation of Neisseria gonorrhoeae from genital and rectal swabs for gonorrhea diagnosis
?? Cultivation of Haemophilus influenzae and other Haemophilus spp. from CSF, blood, and respiratory specimens in meningitis or otitis media cases
?? Recovery of N. meningitidis from cerebrospinal and nasopharyngeal samples for meningococcal disease surveillance
?? Support for subcultures, antimicrobial testing, and quality control of fastidious pathogens per CLSI M100 and ATCC protocols
Key Features:
?? IsoVitaleX Enrichment enhances growth of >90% of N. gonorrhoeae strains within 18-24 hours, outperforming basic blood agar bases
?? Hemoglobin lysis provides essential X-factor without the need for separate supplements; cornstarch detoxifies fatty acids
?? Buffered phosphate system and enriched peptones promote luxuriant, characteristic colonies (e.g., small, pearly for H. influenzae) under CO?-enriched atmosphere
?? Validated for ATCC strains like N. gonorrhoeae 43069 and H. influenzae 10211 with ?70% recovery; suitable for direct plating or swab inoculation
?? Opaque, chocolate-brown appearance with smooth, moist surface; minimizes contamination risk through unique processing
Composition (per liter):
Pancreatic Digest of Casein � 7.5 g
Selected Meat Peptone � 7.5 g
Sodium Chloride (NaCl) � 5.0 g
Corn Starch � 1.0 g
Dipotassium Phosphate (K?HPO?) � 4.0 g
Monopotassium Phosphate (KH?PO?) � 1.0 g
Agar � 12.0 g
Hemoglobin � 10.0 g
IsoVitaleX Enrichment � 12.5 mL
Final pH: 7.2 � 0.2 at 25�C
Preparation:
Suspend 38.0 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 10 g sterile hemoglobin powder (or equivalent 2% hemoglobin solution) and 12.5 mL rehydrated IsoVitaleX Enrichment (one vial per liter, reconstituted in sterile water). Mix thoroughly to avoid bubbles and pour into sterile Petri plates (20-25 mL per plate). Allow to solidify at room temperature; dry the surface briefly if excess moisture is present. Warm plates to room temperature before inoculation. Streak specimens in a "Z" pattern or roll swabs directly; incubate at 35 � 2�C in 3-5% CO? for 18-48 hours. Prepared medium should appear chocolate-brown, opaque, and smooth.
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 the dark, in sealed sleeves or plastic bags; use within the labeled shelf life (up to 112 days). Avoid freezing, overheating, or light exposure; discard if contaminated, discolored, dried, cracked, or excessively moist.
?? 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 for potentially infectious specimens (e.g., hepatitis, HIV)�wear gloves, lab coat, and eye protection. Autoclave all waste, plates, and materials before disposal. If surface moisture is high, air-dry inverted over an offset lid before incubation to prevent sealing. Test medium performance with QC organisms prior to use; suboptimal CO? may inhibit growth.