Overview:
Charcoal Agar Base is a nutritionally enriched, selective solid medium developed by Mishulow et al. in 1953 as an improved alternative to Bordet-Gengou Agar for the primary isolation, cultivation, and maintenance of fastidious Bordetella species, particularly B. pertussis (the causative agent of whooping cough), B. parapertussis, B. bronchiseptica, and B. avium. This charcoal-supplemented base neutralizes toxic fatty acids and peroxides that inhibit growth, providing essential peptides, vitamins, and carbohydrates via HM infusion, peptone, and yeast extract. It is widely used in clinical microbiology for nasopharyngeal swab cultures and vaccine production, often supplemented with 10% defibrinated blood and cephalexin (e.g., Bordet Selective Supplement) to enhance selectivity against contaminants like Staphylococcus and Klebsiella. Incubation at 35-37�C for 3-6 days under aerobic conditions yields small (1-3 mm), grayish-white, shiny, non-hemolytic colonies; confirmation via direct fluorescent antibody (DFA) or biochemical tests.
Applications:
?? Primary isolation of Bordetella pertussis from clinical nasopharyngeal swabs for whooping cough diagnosis per CLSI and CDC guidelines
?? Cultivation and stock maintenance of Bordetella species in vaccine production and research
?? Selective recovery of Bordetella parapertussis and B. bronchiseptica from respiratory specimens
?? Conversion to Chocolate Charcoal Agar (with blood heating) for isolation of Haemophilus influenzae and other fastidious Haemophilus spp.
Key Features:
?? Activated charcoal and soluble starch detoxify inhibitory fatty acids and peroxides, supporting >80% recovery of stressed Bordetella ATCC strains like B. pertussis 8467
?? Enriched base promotes luxuriant growth without overgrowth of contaminants when supplemented with blood and cephalexin
?? Opaque, black medium with undissolved charcoal particles; characteristic small, pearl-like colonies under 35�C incubation for presumptive ID
?? Validated in Manual of Clinical Microbiology (11th Ed.) and APHA standards; superior to potato-based media for consistency in vaccine workflows
?? Versatile for enrichment cultures; inhibits Gram-positive/negative flora (e.g., S. aureus, K. pneumoniae) at high inocula
Composition (per liter):
HM Infusion B (from 500 g equivalent) � 12.0 g
Peptone � 10.0 g
Yeast Extract � 3.5 g
Soluble Starch � 10.0 g
Activated Charcoal � 4.0 g
Sodium Chloride (NaCl) � 5.0 g
Agar � 18.0 g
Final pH: 7.3 � 0.2 at 25�C
Preparation:
Suspend 62.5 g of the dehydrated medium in 1 liter of purified/distilled water. Heat to boiling with frequent stirring 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 blood (10%) and the rehydrated Bordet Selective Supplement (one vial per 500 mL, containing cephalexin 50 mg). Mix thoroughly and pour into sterile Petri plates (15-20 mL per plate). Allow to solidify at room temperature. For Haemophilus isolation, heat the blood-supplemented medium at 80�C for 10 minutes to prepare Chocolate Charcoal Agar. Inoculate nasopharyngeal swabs directly or via enrichment; incubate at 35-37�C aerobically with 60-70% humidity for up to 6 days. Prepared plates should appear black, opaque, and homogeneous with fine charcoal particles.
Storage:
Dehydrated medium: Store in a tightly closed container at 10�30�C, protected from moisture, light, and humidity; stable until expiry date (typically 3 years). Reseal immediately after opening to prevent clumping.
Prepared plates: Store at 2�8�C in sealed plastic bags; use within 2-4 weeks. Discard if dehydrated, contaminated, discolored, or overgrown.
?? For laboratory and research use only. Not for diagnostic procedures without proper validation. Not for human or veterinary consumption. Handle under biosafety level 2; wear gloves, lab coat, and eye protection�use Universal Precautions for respiratory specimens. Autoclave all waste before disposal. Cephalexin supplement may vary by manufacturer; test for antibiotic potency. Suboptimal humidity or temperature may inhibit growth.4.4s