286 



The principal species. 



The colonies from the sulfur-chalk denitrifications, which develop on the 

 broth plates are for a part coloured yellow or reddish brown by carotin 1 ), for 

 the greater part, however, colourless. The brown species is a Micrococcus; it 

 liquefies the gelatin and the micrococci differ much in size; the smaller ones are 

 highly motile, but they lose their motility when transferred to broth-agar, whereby 

 their denitrifying power, too, disappears. The yellow species is related to the 

 brown and consists of small very motile rodlets. Here also the same variability. 



The uncoloured colonies are of two types: soft, and tough or slimy. 



All the soft ones liquefy the gelatin on which they grow intensely; sugars 

 are not fermented, no fluorescence; they belong to three classes different by their 

 size: I. Extensive, rapidly growing, strongly denitrifying. 2. Middle sized, less 

 rapidly growing, as strongly denitrifying. These two classes are allied by inter- 

 mediate forms and may be brought to one single species, Bacterium denilrificans. 

 3. Very small and feebly growing, non-denitrifying bacteria, manifestly living at 

 the expense of organic food produced by the other species through chemosynthesis. 



With the pure cultures on an organic medium of the second form, I have 

 succeeded in obtaining very feeble anorganic denitrifications, hence, chemosynthesis. 

 This could, however, only be observed in the quite young cultures that had but 

 for a short time grown on the broth medium. Cultures which have longer than 

 two or three days been in contact with organic food and the air, can no more 

 denitrify with sulfur and chalk, but still very well in saltpetre broth. For demon- 

 strating the anorganic denitrification, test tubes are partly filled with mud, previously 

 deprived of organic matter by keeping the mud under a saltpetre solution. To 

 the mud sulfur and chalk are added and subsequently l% saltpetre; the dissolved 

 oxygen and the germs are removed by boiling; sterilisation is not wanted, as 

 spore-formers with chemosynthesis do not exist. 



Entrance of air is prevented by a hollow glass sphere, well fitting in the 

 tube and floating on the liquid, but this precaution is not necessary. 



With the pure cultures of the soft colonies I could not obtain any evolution 

 of gas in this mixture, they manifestly lose their autrotrophy still sooner than 

 those of the second group. 



The more or less tough, or slimy, or cartilaginous colonies belong all to Bacterium 

 stutzeri, if taking the conception of species in a broad sense; superficially there 

 is a great difference between the colonies of this group. The usual form, which 

 is very remarkable and easily recognisable by the shape of the colonies, has been 

 described in these Proceedings by Professor van Iterson 2 ). Even in the smallest 

 floccules of the sulfur denitrifications some form of B. stutzeri is found, although 

 the soft colonies prevail. But besides, other varieties of B. stutzeri occur, for example 

 such which sligthly liquefy gelatin, or such which are light brown or rose-coloured, 



') This pigment is soluble in CSa and turns blue or violet with concentrated 

 sulfuric acid. 



2 ) Ophoopingsproeven met denitrificeerende bakterien. Acad. of sciences, Amsterdam, 

 July 1902. 



