280 



posit in a closed bottle will after few repetitions give rise to diminuatiun or cessa- 

 tion of the fermentation. 



For the continuation of the culture by inoculating slight quantities of material 

 of a rough fermentation into the same nutrient liquid, two precautions should be 

 taken. First, the inoculation should be done into the medium, freed from air by boi- 

 ling, the bottle being entirely filled with the hot liquid, so that on cooling no air can 

 dissolve. Second, an acidity of less than 7 proves not sufficient, hence this should be 

 8 or 10 cc., as otherwise the lactic acid ferments might prevail and supplant the 

 sarcine. 



From the necessity of expelling the air we see that the fermentation sarcine 

 undoubtedly belongs to the ordinary anaerobics, which, considering the success of the 

 rough accumulation experiment with aeration, might perhaps not have been expec- 

 ted; but the fact holds good in the same way for the butyric acid ferment, generally 

 accepted as an obligative anaerobic, so that, also with respect to the fermentation 

 sarcine, there should be spoken of microaerophily. Further examination shows 

 that in deep test-tubes with maltwort-agar, very easily pure cultures may be obtained, 

 whereby the sarcine is recognisable by the obvious size the remarkably rapid deve- 

 lopment of its colonies. On the other hand, on maltwort, or broth-bouillon-glucose- 

 agar-plates with or without acid at 37 C., with access of air, no growth at all of the 

 sarcine takes place, as might be expected. Of course the packages can also be seen 

 on the plates without growing and be removed in a pure condition. When we make 

 use of little acid for the rough accumulation, colonies of lactic acid ferments, belon- 

 ging to the physiological genus Lactobacillus, will develop on the plates at the air, 

 which can grow as well with as without air, but whose other life conditions corres- 

 pond to those of the sarcine. In this case the experiment shows at the same time 

 that everywhere in garden soil real lactic acid ferments are present, whereof the 

 proof had not been given until now. 



When using much acid, for example 10 cc. or more normal acid per loocc. of 

 culture fluid, through which the vital functions of the sarcine, such as rapidity of 

 growth and the faculty of assimilating oxygen, are lessened, certain alcohol fer- 

 ments, proper to garden soil, come to development, but they can, together with some 

 of the other impurifications of the rough accumulations, as moulds, Mucor and 

 Oid'ntm, be checked and expelled by exclusion of air, hence, by culture in closed bott- 

 les. To this end however, it is necessary to render the conditions for the sarcine as 

 favorable as possible and not allow a temperature below 37 C. 



The staying out of the butyric acid fermentation (caused by Graindobacter sac- 

 charobutyricum) , which so readily originates with exclusion of air in glucose-bouil- 

 lon and maltwort, is due to the acidity of about 8 cc. or more, whereby this fermen- 

 tation becomes impossible. 



Although it is evident from the foregoing, that the growth of the sarcine is 



less inhibited by the acid than that of the lactobacilli and of the butyric ferment, it 



may still be easily proved that already 7 cc. acid per 100 cc., are less favorable than 



1 or 5 cc., also for the development of the sarcine itself, so that the higher amount 



acid in the accumulation only serves to render competition with the said ferments 



po< Tf by timely transports into maltwort with more than 8 cc. phosphoric acid, 



or by separation in solids, real pure cultures are at disposal, the further transfers, 



