EDWARD C. ROSENOW 579 



METHODS OF STUDY 



By varying the amount of inoculum, tall columns of ascites-dextrose agar and 

 broth were found especially useful in the isolation of streptococci from the joint exu- 

 dates in cases of rheumatic fever and, somewhat later, for the isolation of bacteria 

 from emulsions of tissues and the sediment of laked blood even in diseases generally 

 thought to be due to causes other than infection.' The living cells in the inoculum ab- 

 sorbed oxygen and thus afforded a gradient of oxygen tension. 



The importance of partial-tension cultures in the isolation of pathogens has since 

 been emphasized, especially by Wherry and Oliver.^ In the earlier work on elective 

 localization inoculations in these media sufficed to permit highly sensitive organisms 

 to multiply because the inoculum usually consisted of emulsions of excised tissues 

 containing the metastatic lesions, the sediment of laked blood, the material expressed 

 from tonsils, or pus aspirated from pyorrhea pockets. In later experiments the 

 amount of inoculum was often too small and the bacteria or living cells too few (es- 

 pecially in the case of cultures from devitalized teeth)-' to lower the oxygen tension 

 sufficiently in these media to promote growth of the more strictly anaerobic organ- 

 isms. The addition of fresh sterile tissue is not well adapted for routine work because 

 it is so difficult to exclude contamination. In searching for an inexpensive and easily 

 prepared medium that could be autoclaved and that would furnish a gradient of oxy- 

 gen tension, and other conditions favorable for growth of highly sensitive organisms, it 

 occurred to me that since anaerobes grow readily in brain-mash medium, the addition 

 of pieces of brain to the bottom of tall columns of glucose broth and agar might suffice 

 to lower the oxygen tension sufficiently to permit anaerobes to multiply in instances 

 in which the inoculum must needs be small and free or relatively free from cells or 

 saprophytic contaminants. 



It was found that two or three pieces of brain approximating i cc. each, when 

 added before sterilization to tall columns (8-10 cm.) of glucose broth (0.2 per cent 

 glucose) or agar, contained in test tubes 1.5 cm. in diameter, sufficed after steriliza- 

 tion to decolorize methylene blue in the deeper part of the tubes and keep it so for a 

 long time on standing, and that anaerobic streptococci, tetanus spores, Bacillus 

 welchii, and Bacillus f us if ormis in mixed cultures, and other strictly anaerobic organ- 

 isms grew readily when inoculated into the bottom of the tubes containing the pieces 

 of brain. On the basis of these observations the brain-containing media were adopted 

 in most subsequent work on elective localization. In special instances ascites fluid or 

 serum or fresh sterile tissue is added and the surface covered with a layer of vaseline 

 or paraffin oil; prolonged boiling just before use, to drive off oxygen, is also carried 

 out. 



The successful isolation of the causative organisms of various diseases from tonsils, 

 nasopharynx, cervix, prostate, and especially the periapical tissues of pulpless teeth, 

 usually containing a mixture of bacteria, has often been directly attributable to the 

 use of these media. It has happened repeatedly that not only did growth occur in 



' Rosenow, E. C: J.A.M.A., 63, 903. 1914. 



^Wherry, W. B., and Oliver, W. W.: /. Infect. Dis., 19, 28S. igi6; 20, 28. IQ17. 



■'Rosenow, E. C: ./. Deiil. Research, i, 205. igig. 



