376 STUDIES IN GENERAL PHYSIOLOGY 
at a temperature of 22° C., in the entire absence of oxygen. 
This looks, indeed, as if the energy for a certain small num- 
ber of beats could be furnished through hydrolysis alone, but 
that for a larger number of heart-beats (about 120) the energy 
is obtained, in the presence of oxygen, through oxidation. 
II. ON THE METHOD OF THE EXPERIMENTS 
In the following experiments on the effects of lack of 
oxygen, the oxygen was always displaced by hydrogen; the 
latter was freshly prepared from zinc and sulphuric acid for 
every experiment. The gas was washed thoroughly through 
two bottles of potassium hydroxide, one of potassium per- 
manganate, and one of water. The hydrogen obtained in this 
way was entirely odorless. Before the experiment was begun, 
all air was driven out of the apparatus. The animals ex- 
perimented upon were kept in an Engelmann chamber which 
permitted of direct microscopic observation. 
In this method one fact is to be emphasized, which I have 
not seen mentioned elsewhere. When the living tissue upon 
which we wish to test the effects of lack of oxygen is placed 
in the Engelmann chamber, our judgment of the results is 
subject to the criticism that we do not know the exact 
moment at which the oxygen is all driven out of the liv- 
ing specimen. The resistances to the diffusion of oxygen 
out of the protoplasm are very great, and it may take con- 
siderable time before all the oxygen is removed. As long 
as we find that the phenomena whose dependence upon 
oxygen we wish to study cease immediately after the 
hydrogen is passed through the gas-chamber, this diffi- 
culty is less marked; for even though in this case not all 
of the oxygen had been driven out of the organism, it 
would show only that the particular function which is being 
studied already ceases at a diminution of the oxygen supply, 
and still more so in the total absence of oxygen. It is 
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