362 
Kauffman. — A Contribution to the 
Saprolegnia for purposes of classification. ( d ) The details of technique are 
not especially emphasized by Klebs, and this made it difficult to repeat his 
experiments with ease and rapidity. It was therefore found to be necessary 
to work out in detail the technique used, and it seems advisable to report 
the same, and so make pure cultures of the fungus more available for class 
use or for purposes of research. 
Most of the experiments were done during the winter months of the 
last five years in the intervals of other work. I am indebted to Professor 
R. A. Harper, of the University of Wisconsin, who first suggested to me the 
line of work which has culminated in this paper. My thanks are also due 
to Professor G. F. Atkinson, of Cornell University, and to Professor F. C. 
Newcombe, of this laboratory, for their continued interest and assistance. 
Technique. 
The methods of culture used by De Bary, Humphrey, and others 
usually afford material for study which is fairly reliable, as it can be 
obtained approximately free from bacteria and other fungi. No cultures 
had, however, been made from a single isolated zoospore or gemma up to 
the time of Klebs’s paper, and no method has as yet been outlined for 
cultures with single zoospores. In only one instance have I come across 
a reference to a possible method of zoospore culture. Trow (’95) isolated 
a single zoospore of Aphanomyces by means of cover-glasses on which 
white of egg had coagulated in thin coatings, and which had been floated 
on water containing Saprolegnia , & c. A single culture resulted. On the 
other hand, cultures have been obtained from masses of zoospores pre- 
sumably originating from a single sporange (Maurizio, ’96) ; but in all 
such cases the possibility of contamination with zoospores from other 
species is great. 
Maurizio (’94) isolated the various species by snipping off the ends 
under a lens before the formation of sporanges. From a bundle of hyphae 
cut off in this way and floated in water a single hypha was removed by draw- 
ing up into a pipette and transferring to a fly’s leg on a slide. The water 
on the slide was changed every day by the use of filter paper. If this did 
not yield a single species the process was repeated. His substratum during 
this study were meal-worms, flies, and cress-seedlings. In 1896 he made 
a considerable variety of cultures on peptone, cane sugar, milk sugar, egg 
albumen, Liebig’s extract, beef broth, and glycerine, all of which flourished. 
Boric and salicylic acid were used to keep off the bacteria, and this indi- 
cates that his work was not always done under sterile conditions. Trow 
(’95) used the same method of isolating hyphae, usually selecting such as 
had oogonia on them. 
Klebs (’99) isolated a single gemma of Saprolegnia mixta and under 
entirely sterile conditions succeeded in studying the development of a single 
