Bordet Gengou Agar Plate w/ 25% Sheep Blood

Principle And Interpretation

Bordet Gengou Agar Media were originally formulated by Bordet and Gengou (1) for cultivation of Bordetella species Bordetella pertussis is the causative agent of whooping cough and with the help of cough-plate technique, B.pertussis can be isolated from pharyngeal extracts, nasopharyngeal secretions and pre-nasal swabs. Kendrick and Eldering (2) modified the original media by replacing 50% human or rabbit blood with 15% sheep blood to make the medium more enriched for detection of B.pertussis by the virtue of its haemolytic reaction. Enrichment of the basal media with 25% human blood aids in the detection of Mycobacterium species from small sputum inocula and in Streptomycin sensitivity testing (3). The medium is highly nutritious thus supports luxuriant growth of Bordetella species and can also be used for mass cultivation of B.pertussis for vaccine production (4) and for maintaining stock cultures (1). Potato infusion and peptone serve as carbon and nitrogen source, amino acids while glycerol and blood enrichment provides additional nutrients. Sodium chloride maintains osmotic equilibrium. Incubation must be carried out in a moist chamber (60% humidity) at 37°C for upto 7 days. Medium must not be over dried before use. After 40 hours B.pertussis colonies appear smooth, raised, glistening with a zone of haemolysis. Some strains of Bordetella are not haemolytic. For confirmation, serodiagnosis and biochemical test must be performed. For isolation of B.pertussis from specimens, use standard procedures. Incubate the plates in a moist chamber at 35-37°C for 7 days and examine daily with or without dissecting microscope (oblique illumination) to detect the presence of B. pertussis . Sometimes the accompanying mold colonies can mask the B. pertussis colonies. Use sterile scalpel or needle to remove the portion of the agar that contains spreading colonies of moulds. B. pertussis colonies may not be visible without the aid of a microscope for 2-4 days. After 7 days of incubation plates must be discarded as negative. Some Haemophilus species will grow on Bordetella isolation media and cross-react with B. pertussis antisera. It may be prudent to rule out X and V factor dependence.