96 DIFGOMANUAL 



Hemophilus pertussis in the diagnosis of whooping cough. This is a modification 

 of the medium originally described by Bordet and Gengou^ in 1906 for the culti- 

 vation of H. pertussis and is prepared according to the formula recommended 

 in Diagnostic Procedures and Reagents^ of the American Public Health Associa- 

 tion for the isolation of this organism. The addition of 1 per cent Proteose 

 Peptone to the medium is suggested if employed for mass culture of H. pertussis 

 as in vaccine production. 



The "cough plate" method for the diagnosis of whooping cough was originally 

 reported by Chievitz and Meyer^ in 1916. Lawson and Mueller^ in 1927 and 

 Sauer and Hambrecht^ in 1930 used modifications of the Bordet Gengou Medium 

 to demonstrate the value of the cultural diagnosis of this disease. This method 

 has been applied routinely as a diagnostic procedure for public health labora- 

 tories as a result of the thorough and painstaking investigations of Kendrick and 

 her associates. Kendrick and Eldering^ first used a modified Bordet Gengou 

 Medium for the isolation and propagation of H. pertussis. Eldering and Ken- 

 drick'^ reported that the addition of 1 per cent of Proteose Peptone or Neopep- 

 tone increased the growth of H. pertussis thereby increasing the yield for vaccine. 



With this modification of the Bordet Gengou Medium, enriched with 15 to 20 

 per cent blood, the appearance of colonies of H. pertussis is typical, being 

 smooth, raised, glistening and not over 1 mm. in diameter. They are of a pearly, 

 almost transparent appearance, and are surrounded by a characteristic zone of 

 hemolysis which is not sharply defined, but which merges diffusely into the 

 medium. The zone of hemolysis usually is absent if 30 per cent or more blood is 

 added to the medium. Sterile sheep, rabbit or human blood may be used in pre- 

 paring the medium. Horse blood should not be used in preparing vaccine. 



Kendrick, Miller and Lawson'^ and Kendrick, Lawson and Miller^ recom- 

 mended that, after exposure, cough plates prepared from the modified Bordet 

 Gengou Blood Agar should be incubated at 37°C. During the first 48 hours 

 incubation they are examined for contamination by molds and spreaders, which 

 are cut aseptically from the medium. The plates are then examined twice daily, 

 using a hand lens, until typical colonies of H. pertussis are found or until dis- 

 carded after 6 days of incubation. 



Maclean^ used Bacto-Bordet Gengou Agar Base and reported it to be efficient 

 in the isolation of H. pertussis. He further reported that this medium was a 

 valuable standard for the comparison of various lots of media prepared from 

 ingredients. 



Tarshis and Frisch^o investigated the addition of bank blood to various media 

 for the cultivation of tubercle bacilli in pure culture and directly from sputa 

 under routine diagnostic conditions. Three standard tuberculosis media were 

 used in the comparative study. They recommended the addition of 25 per cent 

 bank blood to Bacto-Bordet Gengou Agar Base or Bacto-Blood Agar Base with 

 1 per cent glycerol added since media of this type grew tubercle bacilli from 

 small inocula producing colonies that were readily recognized. These media 

 were easily prepared and in addition were economical. They were also satis- 

 factorily employed in streptomycin sensitivity tests. 



To rehydrate the medium, suspend 3 grams of Bacto-Bordet Gengou Agar 

 Base in 100 ml. of a 1 per cent solution of glycerol in distilled water and heat 

 to boiling to dissolve the medium completely. Distribute in flasks and sterilize in 

 the autoclave for 15 minutes at 15 pounds pressure (121°C.). It is cooled to 45 

 to 50°G. and aseptically enriched with 15 to 20 per cent sterile sheep, rabbit, 

 or human blood^ and is poured into sterile petri dishes. The blood should be 

 used when fresh, never more than 72 hours after it has been obtained. Satisfac- 

 tory plates should be bright cherry red in color and free from bubbles and lumps 

 of agar. Plates may be used as long as they remain moist and red, usually two 



