Charcoal Agar Base

         

Specifications

Ingredients Gms / Litre :
Beef heart, infusion from 500.000
Peptic digest of animal tissue 10.000
Yeast extract 3.500
Starch, soluble 10.000
Charcoal 4.000
Sodium chloride 5.000
Agar 18.000
Final pH ( at 25°C)
**Formula adjusted, standardized to suit performance parameters
7.3±0.2


Principle & Interpretation :
The genus Bordetella comprised of 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 for B. pertussis and B.parapertussis, while B.bronchoseptica is found in a large variety of animals and rarely 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 and 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. Charcoal Agar is prepared according to the method of Mishulow, Sharpe and Cohen(2). This medium can be used as a replacement for BordetGengou Agar for isolation of B.pertussis and for the production of B.pertussis vaccines. Charcoal Agar supplemented with hors e 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 repression of unwanted flora during the long incubation period on nutritious media. Penicillin can be added to the medium as an antimicrobial agent for restricting the ot her contaminants. However Penicillin resistant floras still cause contamination, which as observed by Lacey (4). Methicillin was found to be superior than 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)
.
The ingredients like beef heart infusion, peptic digest of animal tissue, yeast extract provide essential nutrients to the or ganisms. 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 obta in a uniform charcoal suspension (7)
.
For isolation of B.perthssis from suspected cow whooping cough collect the nasal swabs in early stage of the illness and place in tubes of half strength Charcoal Agar Base supplemented with 10% v/v lysed defibrinated horse blood and Bordetella Selective Supplement (MS2004). Generously inoculate the swabs on to thick layer of Charcoal Agar Base containing 10% v/v blood and Bordetella Selective Supplement (MS2004). Non-selective medium (without MS2004) may be used in addition. Replace the swab in the original transport medium and hold at room temperature. Incubate the plates at
35°C in a moist atmosphere (60-70% humidity) upto 6 days. Examine plates after 40 hours incubation and twice daily thereafter. Small shiny grayish white, round corner, colonies of Bordetella species are observed on plates. Confirm the findings with DFA i.e. Direct Fluorescent Antibody testing. To make earlier diagnosis, perform direct fluorescent antibody testing on the secretion (8)
.