28J 



arose: wliich are tlie aerobic spore-forming- bacteria, whicli can 

 develop a( temperatures of 15° C. or lower and undei- favorable 

 feeding conditions? We knew already tliat the obtaining of B. 

 inegntherium might give an answer to tlie question, for example in 

 case the spores of this species were only present with those of 

 B. mesentericus, but it seemed possible that free competition with 

 the soil bacteria would exclude B. megatherium and that some other 

 species could appea)'. The chief aim of the experiment was to 

 exclude B. mesentericus, the common hay bacterium, which produces 

 substances very noxious to othei' species, and this is to be leached 

 by the low temperature, as the minimum for the growth of this 

 species is at about 20° C. The simultaneous development of B. 

 megatherium is of less importance as it is innocuous to other kinds. 

 Of course we had to reckon with the butyric-acid and butylic fer- 

 mentiitions, which may very well occur at 15° C, but strong aera- 

 tion prevents them efficiently. 



Although we could expect that the one or more species that were 

 to develop under the chosen conditions would possess a higher 

 temperature optimum than that used by us, we had not to fear a 

 failure if only we cultivated above their minimum. 



Knowing that the spores of some spore-formers, for example those 

 of the butylic ferments, and thus perhaps, too, those of the species 

 we sought for, could not or hardly resist boiling, the heating of 

 the culture liquid containing the inoculation material and wanted 

 for killing the non-spore forming species, was not continued much 

 above 85° or 90° (/.and only for a few seconds. We used flasks 

 half filled with about 30 cM" liquid, and in order not to miss 

 somewhat rarer species, we inoculated with so much soil that on 

 the bottom a layer of about 1 cM precipitated. This soil had 

 previously been well-divided and freed from coarse particles. In 

 such a thick layer a beginning of anaërobiosis is possible, but by 

 shaking, butyric-acid or butylic fermentation may easily be stopped. 



For food we used at first malt- wort, diluted to 2° to 5° Balling, 

 later broth-bouillon with 27o fo 57o cane sugar, or glucose. Addition 

 of chalk is not absolutely wanted for the success of the experiment 

 but its presence proved favorable. 



After we had ascertained with pure cultures of B. polymyxa that 

 ammonium salts, nitrates and asparagine are very good sources of 

 nitrogen, we also accumulated with sugars and ammonium sul- 

 phate, in a solution of tapwaier 100, 2 to 5 7o glucose or cane 

 sugar, 0,05 7, (NH,),SO,. and 0,02 7„ K, HPO, with some chalk. 

 The execution of the experiment is as above, but after pasteurising, 



