(130 5 
After 2 or 3 successive inoculations in the same liquid a constant 
bacterial mixture was obtained. 
I tried to apply these principles to the isolation of denitrifying 
land-bacteria, and so-doing [| succeeded indeed, when using calcium- 
tartrate as source of carbon, to accumulate Bacillus vulpinus, hereafter 
to be discussed. 
It proved however to be a fundamental improvement wholly or 
partly to exclude the access of air as thereby the growth of the 
denitrifying bacteria is not in the least impeded, whilst a number of 
other aerobie bacteria are very much hindered in their development. 
Of the numerous methods of culture under exclusion of air I have 
followed the simplest, namely the “bottle method”, long since 
in use in the Bacteriological Laboratory at Delft for the examination 
of the sulphate reduction by microbes and the lactie-acid fermen- 
tation. For my experiments this method proved perfectly adapted, 
as the quantity of air which finds access, can thereby easily be 
regulated. An ordinary, narrow-mouthed stoppered bottle, with an 
exactly fitting stop, is quite or partly filled with the culture liquid, 
and after sterilising or not, according to circumstances, the bottle is 
placed in the thermostat for culture. 
1. Historical. 
The reduction of nitrates by bacteria constantly begins with the 
formation of nitrite. This may be further converted in five different 
ways, VIZ. : 
1st. It may be reduced to ammonia. 
24, Tt may be converted into unknown, nonvolatile nitrogen 
compounds. 
Zed, Tf in the liquid acid is formed simultaneously, it may give 
rise to the development of nitrogen-oxygen compounds. 
Ab Tt may be decomposed in alkaline solution under formation 
of nitrogen-oxygen compounds. 
5th, The nitrite may, in alkaline solutions, give rise to the devel- 
opment of nitrogen without the production of nitrogen-oxygen com- 
pounds. This is denitrijication proper, of which here is only question. 
Already in 1814 Davy’) states that during putrefaction of aninal 
matter nitrogen as such is freed. “Here it is again seen,” says in 1860 
G. J. Munprr?), from whom I borrow this particular, “if one wishes 
1) Elemente der Agriculturchemie, Berlin 1814, ea: 
2) De Scheikunde der Bouwbare Aarde, 1860, dl. 3, blz. 58. 
Js 
