10 



Coarsely ground rye with some chalk and inoculated with fertile garden soil is 

 mixed with water in a deep beaker to a thick solid paste, boiled during some seconds 

 to kill the non-spore-formers and cultivated at 25 to 30 C. As the spores of B. po- 

 lymyxa soon die at boiling, the heating must last but a short time. After a few days 

 the surface is covered with a coherent film of B. mesentericus l ) and other closely 

 related species, while in the depth a butyric-acid fermentation takes place, usually 

 simultaneously with butylic-alcohol- and polymyxa fermentation. 



It is clear that this accumulation reposes essentially on a temporary anaerobiosis 

 of B. polymyxa, which can also grow aerobic and so behaves like the alcohol yeast and 

 the Aerobacter-Coligroup among the bacteria. The rye produces the sugar causing the 

 fermentation, i.e. the source of energy, which makes the anaerobiosis possible so 

 long as the ..excitation oxygen" is still sufficiently present, albeit chemically non- 

 demonstrable, whereas the want of ,, oxidation oxygen", which is required for aero- 

 biosis in much larger quantity as source of energy, is temporarily excluded. P a s- 

 te u r's statement: ,,la fermentation est la vie sans air" is evidently applicable to 

 B. polymyxa. 



By sowing out the fermenting matter from the depth on wort-agar, ordinarily 

 already after few days the polymyxa colonies become visible as lumps of slime, to- 

 gether with the unavoidable flat spreading colonies of B. mesentericus. 



This method can only produce those varieties of B. polymyxa which are able to 

 resist a relatively high concentration of the food. Another accumulation method by 

 which also forms adapted to a lower concentration of food are obtained is based on the 

 aerobiosis of our bacterium. 



After the observation had been made that flasks of boiled wort, not sufficiently 



sterilised, were not seldom spoiled at the low temperature of 15 C. by the develop- 





 ment of B. megatherium and never by B. mesentericus, whose germs were certainly also 



present, the question arose : which are the aerobic spore-forming bacteria, which can 

 develop at temperatures of 15 C. or lower and under favorable feeding conditions? 

 We knew already that the obtaining of B. megatherium might give an answer to the 

 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 appear. The chief 

 aim of the experiment was to exclude B. mesentericus, the common hay bacterium, 

 which produces substances very noxious to other species, and this is to be reached 

 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 buty- 

 lic fermentations, which may very well occur at 15 C, but strong aeration 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. 



') This film may be colourless, brown, red, and even jet black according to the acciden- 

 tally present varieties of B. mesentericus. The black form is rare and sometimes obtained by 

 the ..mesentericus experiment" with unwashed currants (boiling with chalk, cultivating at 

 aeration at 30 to 40 C.). 



