315 



If such a culture itiass, from wliich the free oxygen is completely withdrawu, is 

 contained in a deep experiment-tube, where the air can only find access from above, 

 then, if the growth is favoured by a certain oxygen tensiun, there must result at the 

 very place where this tension becomes optimum, an opaque and distinctly visible 

 viveait of colonies, which are greater than the colunies beneath and above this niveau. 



The easiest way for completely removing the oxygen, is to sow simultaneously 

 an aërophilous species, not acting injuriously on the development nor disturbing the 

 observation of the anaërobic. Such an aerobic must have the following qualities: 

 The oxygen must be completely absorbed, without exciting so much growth in the 

 surface of the culture mass, that the colonies of the anaërobic become indistiiict. Be- 

 sides, an easy recognition in the microscopic preparation, and a simple separation of 

 the aerobic and the anaërobic must be possible. 



In trying various species of microbes, I found some kinds of yeast to be most 

 efficiënt for the research of the anaërobics of putrefaction and of sulphate reduction, 

 as for these processes no carbohydrates are essential, in which case yeast does not 

 grow strongly, whilst it is distinctly recognisable under the microscope. Besides, yeast 

 can easily be separated from the anaërobics of proteïne putrefaction, because it dies 

 at 50" a 60» C, whilst the spores of the latter can be heated to 90" a 100" C. without 

 dying. For the examination of those anaërobics which require sugar in their food, as 

 for instance the butyric ferments, it is preferable, for oxygen absorption, to make use 

 of certain blastomycetes (which grow and reproduce like yeast, whilst alkohol fermen- 

 tation is absent) or aerobic bacteria, which don't produce acid, nor liquefy gelatine. 

 Good results were obtained with a red blastomycete, isolated from garden-soil, and 

 with Bacillus fliiorescens non Uquefaciens. 



It is good (but not always necessary), to place the prepared experiment-tubes, 

 in an exsiccator which is vacuated. For this vacuation a Körting-waterjetpump with 

 manometer will suffice, by which at the same time the pression of the gas used may be 

 measured. 



Another very suitable method to state the influence of oxygen on the growth, is 

 to cultivate in the »hunii(l room« on the objectbearer under the cover-glass, in a not 

 too small quantity of the nutritious liquid, but in such a way as to keep the prepara- 

 tion thin enough for the microscope. In this way it is possible to observe, in the same 

 preparation, first the figure of respiration and afterwards the growth. 



The species of obligatous anaërobics which I have examined are the following. 



Butyricferment (Granulobacter saccharobutyriciim). This anaërobic is extremely 

 common in garden-soil. Fit material for figures of respiration is to be obtained as 

 follows. Water with some kalium phosphate and magnesium sulphate and 5 or lOpCt. 

 glucose is boiled in a little flask with so much fibrine that a thick paste is formed. 

 During the boiling an infection with garden-soil is practised, in which only spores of 

 bacteria remain alive. In the thermostate a vegetation of aerobics develops first, 

 which, by the absorption of the oxygen, introduces butyric fermentation. Sometimes 

 this fermentation will follow, even in absence of aerobics, i. e. notwithstandig the 

 entrance of air into the mass of fibrine, thus showing that some aëration is certainly no 

 bar to this process. If perhaps an aerobic grows too strongly, reinfection in another 

 flask with the same mixture, will suffice to make it disappear and at length still to 

 obtain an almost pure butyric fermentation. If in the infection material there are too 



