MR. A. J. EWART ON ASSIMILATORY INHIBITION. 865 
method. The second is the reduced indigo test, first brought 
into prominence by Regnault and subsequently by Beyerinck *. 
This method is, however, unsatisfactory, because it requires the 
presence of a slight excess of sulphuretted hydrogen or NaHSO, 
in the reduced indigo solution; and when once the latter has 
become blue, it can only be reduced again by treatment with 
fresh SH, or NaHSO,. The poisonous action of the sulphuretted 
hydrogen, and the fact that once the colour returus the experi- 
ment ceases, continuous observations being possible only by 
adding fresh reduced indigo solution, form serious objections to 
the method. The last objection also applies to the third method, 
which has indeed found but little practical application, of using 
the conversion of reduced hemoglobin into oxyhemoglobin as a 
test for the evolution of oxygen. 
The advantages of the Bacterium method are that it is much 
more delicate and easier to observe, the injurious influence of the 
Bacteria is almost nil, continuous observations are possible with 
the same preparations, and, lastly, the rapidity of the movement 
and number of moving Bacteria enable a relative estimation to 
be made of the amount of oxygen evolved, and hence of the 
intensity of assimilation. “Throughout the investigation the 
Bacterium method was used as a test for the evolution or non- 
evolution of oxygen. The Bacterium employed was Cohn's 
Bacterium Termo (= Proteus vulgaris, Hauser; = Bacillus lique- 
faciens vulgaris, Beyerinck), slope or stab cultures being made 
on Bouillon-Agar, and allowed to develop at a temperature of 
25°C. In actual use the cultures should never be more than 
1 to 2 weeks old, and the Bacteria should be taken from the 
edges of the growth, where it is of more recent formation, and 
hence consists almost entirely of motile forms. 
Great care must always be taken to ensure the absolute purity 
of the cultures and their entire freedom from anærobic forms. 
This is readily ascertained by mounting the Bacteria in a drop 
of sterilized nutrient solution, and, after making certain of the 
absence of all air-bubbles, riuging the cover-slip with either pure 
vaseline, or vaseline stiffened with a little wax or paraffin, ac- 
cording to the temperature of the room. 
If the culture is a pure one, the movement of the Bacteria 
ceases as soon as the enclosed dissolved oxygen is exhausted, and 
if any Baeteria continue to move after haviug been for one hour 
* Beyerinck, in Bot. Zeit. 1890, p. 742. 
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