Mar. 3,1923 
Gray Mold of Castor Bean 
693 
coated cover slip, ejection being stimulated for the moment by a slight 
touch on the edge of the desk; (b) a segment cut from the side of the 
disk and crushed in a drop of sterile water, the resulting suspension of 
spores being then applied directly to the agar surface of a cover slip with 
a platinum loop; (c) spores scraped from the side of the tube to which 
they had been shot when the apothecium was first mature; these were 
applied as in (b). 
In each case, the cover glass was then placed in position over a Van 
Tieghem cell which had previously been made ready by ringing above 
and below with sterile vaseline, sealing to a sterile slide, and pouring in 
a few drops of sterile water. A sterile moist chamber was thus secured 
which could be moved about as much as desired without fear of con¬ 
taminating the contents. Several ascospores were observed, singly and 
in groups, and their germination and development were watched with the 
higher powers of the microscope. All were practically in one plane so 
that it was easy to mark with India ink the position of ger min ating single 
spores. After a few hours several such single ascospore strains were cut 
out and removed to a second agar-coated coverglass and cell, where 
their purity was verified and further development watched. The trans¬ 
fer was accomplished by lifting the coverglass between the thumb 
and forefinger of the left hand and cutting out the area marked, a milli¬ 
meter or so square, with a No. 24 platinum-wire needle which had a very 
thin sharp, knife-like point. Later these single spore strains were trans¬ 
ferred to tubes of agar. 
Inasmuch as this method of isolating single spores is not used as widely 
as its practicability would justify, the writer wishes to call attention to 
it as a simple, quick, and reliable means of obtaining single spores apart 
from one another and from all contaminations, especially useful when 
the spores are exceedingly small. It was developed and used by the 
writer at Iowa State College in 1916-17. 7 Later Durrell (8) used the 
same method for making single spore isolations. Many workers have 
used the Van Tieghem cell for isolating single spores of fungi, as, for 
example, Sherbakoff in his work on the genus Fusarium {12, p. 102 - 
104), in which he emphasizes the importance of obtaining single spore 
strains and the difficulties often attendant thereupon; and most note¬ 
worthy of all, Emil Christopher Hansen who as long ago as the middle 
of the nineteenth century used a method very similar to the one here de¬ 
scribed for securing single yeast cells in fermentation technic (ri, p. 
106 ,107). But insofar as the present writer has been able to determine, 
precisely this method has not previously been described. When spores 
are large enough to be seen with the low power through the bottom of a 
Petri dish and contaminations are not a serious factor, this method would 
not be justified, of course. 
The several cultures derived from all of these three sources of asco¬ 
spores were typical in every way of the Botrytis cultures obtained from 
conidia. The typical gray mold developed quickly, and this was fol¬ 
lowed shortly after by sclerotia. Inoculations of castor bean plants 
made with these cultures, after the usual method (described on p. 703). 
produced the typical disease. Reisolations made from such diseased 
plants were held and used, along with numerous other cultures, for later 
production of apothecia. 
7 Godfrey, G. H. culturai, studies of leptosphaeria coniothyrium causing raspberry cane 
bught. Thesis, Iowa State College, 1917. (Not published.) 
