11 



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 ma- 

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

 above 85 or 90 C. and only for a few seconds. We used flasks half filled with about 

 30 cm 3 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 anaerobiosis is possible, but by shaking, butyric-acid or butylic fermen- 

 tation may easily be stopped. 



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

 bouillon with 2% to 5% 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 sulphate, in a solution of tapwater 100, 2 to 5% glucose 

 or cane sugar, 0,05% (NH 4 ) 2 SO 4 , and 0,02% K 2 HPO 4 with some chalk. The execution 

 of the experiment is as above, but after pasteurising, the butyric-acid fermentation 



must be more completely excluded than when using broth-bouillon or malt-wort. 



* 

 For although the latter liquids contain an excellent nitrogen food for B. polymyxa, 



they are of less value for the butyric-acid ferments, for which the ammonium salts 

 are preferable. Hence, in this case it is advisable to use a large Erlenmeijer- 

 flask, as the great volume of soil which sinks to the bottom as inoculation material, 

 can then be better aerated, by which butyric fermentation is prevented. 



Although the growth is slow at the low temperature the liquid becomes distinctly 

 turbid and in most cases this is accompanied with fermentation. This fermentation 

 especially awakened our attention as we had expected an accumulation of B. me- 

 gatherium, which causes no fermentation at all. 



As the Coli- and A erogenesier mentations had been prevented by the previous 

 heating, the butyric-acid and butylic fermentations by the aeration, we now expected 

 that the fermentation of B. polymyxa was obtained, and this was confirmed by the 

 pure culture. The fermentation which is chiefly an alcoholic one, proves that our bac- 

 terium belongs to the facultative (temporary) anaerobes, and the examination of the 

 gas showed that it is almost pure carbonic acid. 



One of the most notable qualities of B. polymyxa is its secretion of pectinase, i.e. 

 the enzyme by which some microbes dissolve the central lamellum of plant tissues, 

 thereby disintegrating them into cells. Hence, B. polymyxa like B. mesentericus may 

 under certain circumstances play a part in the retting of flax, although the real agent 

 in this case is the anaerobic B. pectinovorum. 



Beans, peas and other plant seeds, left to spontaneous corruption, may change 

 into rich cultures of B. polymyxa, the cell-walls of cotyledons and of endosperm being 

 easily attacked by pectinase, whereby the interior of the seeds is changed to a pulpous 

 mass !). For the preparation of a pure culture this method is less recommendable 

 than the tw r o foregoing accumulations, on account of the numerous hay bacteria 



!) The enzyme seminase, which changes the endosperm of the Leguminosae (Indigofera, 

 Ceratonia) into mannose, is perhaps identic with the pectinase of B. polymyxa. 



