CLASSIFICATION OF ANAEROBIC BACTERIA 547 



Probably tissue-containing media bes^ fulfill the above require- 

 ments. They need no more glucose than that which they derive 

 from the tissue contained in them. Brain medium as used by 

 von Hibler would be excellent were it made up with sufficient 

 liquid for titration purposes. Von Hibler titrated liquid from 

 his brain medium with litmus against potassium hydrate or 

 hydrochloric acid. Today workers would prefer to use a hydro- 

 gen-ion determination, for which a fairly clear liquid is neces- 

 sary in case the simple colorimetric method is employed. Brom- 

 thymol-blue is a suitable indicator for making this separation. 

 At present most laboratories use the beef-heart medium intro- 

 duced by Robertson and Martin for anaerobic study because it 

 has many technical advantages over brain medium. It should 

 preferably contain about 5 per cent of peptic digest broth and 

 should be made with twice its weight of distilled water and tubed 

 in large tubes in order that enough liquid may be present for 

 hydrogen-ion determinations on several occasions. 



Ten days' incubation at 37° will be found quite sufficient in 

 most cases for such a determination as we wish to make. Von 

 Hibler found the reaction decidedly acid or alkaline in brain 

 medium after five days. But to allow time for weakly proteo- 

 lytic organisms which form acid from glucose, to carry the 

 reaction over to the alkaline side, we should incubate the cul- 

 tures for twenty days at least. Such organisms do not at first 

 produce gross signs of putrefaction, but their proteolytic tenden- 

 cies may be tested for by the lead acetate test for hydrogen 

 sulfide. There may be soil anaerobes which do not grow at 37°. 

 Time and temperature for incubation will have to be decided 

 upon for such organisms separately in case they are found. 



Von Hibler (1908, p. 88) found that with his pasty brain 

 medium the mode of incubation, aerobic, or anaerobic in hydro- 

 gen or in carbon dioxide, made no difference in the reaction. 

 But if we are to use a medium with a considerable amount of 

 liquid on the surface it would probably be unwise, even though 

 anaerobes grow in the medium with the surface open to the air, 

 thus to incubate our organisms for twenty days. An an'aerobic 

 method should be employed. The simplest method available 



