364 Kauffman . — A Contribution to the 
a cold window-sill or in a refrigerator at a temperature between 5 0 and 
io° C. or lower. The mycelium immediately begins to grow vigorously, 
while the bacteria multiply slowly, so that in from twelve to eighteen hours 
the margin of the mycelial mass outstrips the bacteria by as much as 
4-6 mm. From the outer zone a piece of gelatine containing the ends of 
hyphae is cut out with a slender scalpel, and transferred to a clean dish, 
where it will again grow rapidly. Usually this is sufficient to separate the 
bacteria ; if there are still some present the process is repeated. The new 
growth may appear to contain bacteria because of the action of proteolytic 
enzymes which soften the gelatine around the new culture ; this appear- 
ance is still more exaggerated by the use of some commercial extracts of 
beef whose transformations produce a turbid liquid. It may be added that 
no lactic acid or other antiseptic was used, although the mycelium grows 
in acidified solutions ; it seemed desirable that no stimuli of such a kind 
should be present before the regular experiments were begun. 
If now there is only one species present, it is easy to obtain a culture 
from a single zoospore, and even if several species are present one of the 
number can be easily separated. A piece from the margin of the Petrie 
dish culture which is free from bacteria is transferred into sterile water 
in a small glass capsule, where, after twenty-four hours, it will be found to 
have produced sporanges and zoospores abundantly. The spores are more 
active and escape more readily from the sporange if the capsule is placed 
in a warm place a short time before using. The water is found to be 
swarming with spores and can be taken up in a sterile pipette and a drop 
of it mixed with 20 cc. of sterile water ; from this diluted fluid a pipette- 
ful is again taken and squirted over the surface of a clean gelatine plate 
in as fine droplets as possible. After another twenty-four hours or less 
one can find germinated spores lying far from any other spores, and these 
are then cut out with a very narrow and pointed scalpel and transferred to 
a gelatine dish as the final step in separation. We now have a culture 
from a single zoospore, and all further experiments can be made on my- 
celium obtained from it. 
As all the species which were studied produced so-called gemmae, 
these may be used in the manner of Klebs and Trow as the starting-point 
of cultures. Their method, however, requires more skill and is to be 
preferred only where it seems difficult to get zoospores. It can also be 
used where it is desired to get a definite species from a mixture of several 
species. As a last resort, the method outlined by Maurizio is available. 
The results of the present paper were obtained from cultures which 
were started from single spores. After the isolation was accomplished, the 
work was done with scrupulous care as regards sterilizing the instruments, 
glassware, and solutions used. It is very easy to transfer accidentally spores 
or bits of mycelium from one set of cultures to another, and hence mix up 
