Charcoal Blood Agar Base, HiVeg™

Principle And Interpretation

The genus Bordetella contains four species: Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica and Bordetella avium (1). Genetic studies have shown that these organisms are very closely related to each other. Humans are the only host of B. pertussis and B.parapertussis, while B.bronchoseptica is found in a wide variety of animals and occasionally found in humans (2). B.avium is found in birds. Bordetella species are obligately aerobic and metabolically not very active. They are non-motile except B.bronchoseptica. B.pertussis is the major cause of whooping cough or pertussis. B.parapertussis is associated with a milder form of the disease (3). Primary isolation of B.pertussis in particular, requires the addition of charcoal, 15-20% blood to neutralize the growth-inhibiting effects. Isolation of this organism requires enrichment medium.

Charcoal Agar is prepared according to the method of Mishulow, Sharpe and Cohen (2). This medium can be used as a replacement for Bordet-Gengou Agar for isolation of B.pertussis and for the production of B.pertussis vaccines. Charcoal Agar supplemented with horse blood can also be used for the cultivation and isolation of Haemophilus influenzae (4). The difficulty in the isolation of Bordetella pertussis from nasopharyngeal secretions is the inhibition of associated flora during the long incubation period on nutritious media. Penicillin is added to the medium as an antimicrobial agent for restricting the other contaminants. However Penicillin resistant floras still cause contamination that was observed by Lacey (4). He therefore supplemented penicillin with diamidino-diphenylamine dihydrochloride, thereby increasing the selectivity of the medium. Methicillin was found to be superior to Penicillin in suppressing unwanted nasopharyngeal flora as observed by Broome et.al. (5). Sutcliffe and Abbott found that Cephalexin was still better than Methicillin (6). Regan and Lowe (7) have further showed that Charcoal Blood Agar Base of half strength with cephalexin is an excellent selective enrichment transport medium. Cephalexin is added to inhibit contaminant gram-positive organisms that may be present in specimen. Both non-selective and selective media should be inoculated since some B.pertussis strains may be slightly inhibited by cephalexin. Charcoal Blood Agar Base, HiVeg™ is prepared by using vegetable peptones in place of animal based peptones which make the media free of BSE/TSE risks. Charcoal Blood Agar Base, HiVeg™ is used for the cultivation of B.pertussis for vaccine production. Medium ingredients like HiVeg™ peptone, HiVeg™ extract and yeast extract provide essential nutrients to the organisms. Sodium chloride maintains osmotic balance. Starch soluble and charcoal neutralizes substances toxic to Bordetella species such as fatty acids. Charcoal has the tendency to settle at the bottom of the flask. Therefore, before dispensing, swirl the flasks gently to obtain a uniform charcoal suspension (8). The medium can also be used for the maintenance of stock cultures of Bordetella pertussis on slants with weekly subcultures.