34 
Ewartj Can Isolated Chloroplastids Continue to Assimilate ? 
must be taken to ensure tbat no oxygen can reacli the pre- 
parations from without. K n y aclmits that in unringed preparations 
it is only when a large number of bacteria are enclosed and the 
coverslip is a large one that the bacteria in the centre of the 
field come to rest. The number of bacteria used should, however, 
always be proportion ate to the amount of assimilating tissue which 
is being examined, When very small and minute objects are 
being tested, it is important that comparatively few bacteria 
should be enclosed, so that the movement of the individual 
bacteria can be readily followed. 
Kny’s observation upon the power of Vaseline to retain 
small traces of oxygen, which are slowly given up on warming, 
is an extremely interesting one, but only to be expected from 
the results given on p. 420 of the work on assimilatory Inhibition. 
(Jour. Linn. Soc. V. XXXI.) It was chiefly for ihis reason that 
a thick ringing with a mixture of wax and Vaseline, or parafin 
and vaseline was employed by the author. The method necessarily 
adopted of heating the mixture and applying it in melted form 
ensured that practically the whole of any dissolved oxygen 
present would be driven out. Moreover, the fact that in all 
cases, the preparations were first kept in the darkness until all 
movement of the bacteria had ceased, and that, as a general rule, 
the stoppage and recommencement were repeated several times, 
is a sufficient indication that the ringing employed was in all 
cases an adequate one. 
It is quite true that it is impossible to obtain even pure 
cultures of absolutely monotypic sensibility, but there can be no 
doubt that the use of pure cultures has caused the bacterium 
method to progress one step nearer perfection in this respect. If 
the bacteria from young agar cultures are mixed with a little 
sterilized water in tubes of special shape kept obliquely slanted, 
the most actively motile bacteria collect at the upper surface 
of the fluid, so that a drop taken from the surface is found 
to contain bacteria, all of almost precisely uniform sensitivity. 
It is, however, always of greater importance that the object tested 
should be under as normal conditions as possible, for the 
sensitivity of the bacterial reagent can be tested both before and 
after experimentation. 
With regard to the light intensity employed by Kny, his 
later explanations have shewn that my objections were quite 
unfounded. In my own experiments no measured or absolutely 
constant source of illumination was employed. At Leipzig on 
bright days the light from a N. E. window was used; on dull 
days that from an Auer lamp, so that in both cases, with a fully 
opened iris diaphragm, the light was to the eye of dazzling 
intensity. In the tropics, the light reflected from white clouds 
was preferably employed. When any observations were made the iris 
diaphragm was gradually opened until the movement of the bacteria 
commenced and became fully active in the neighbourhood of the 
assimilating cells. If, however bright the illumination might be, 
