346 
Journal of Agricultural Research 
Vol. XXIV. No. 4 
ISOI^ATION OF CUI^TURES 
The original cultures were all obtaine’d from infected tubers by the 
following method: 
The infected tubers were thoroughly washed, dipped into a i to i,ooo 
solution of mercuric chlorid, and cut open. In order not to contaminate 
the infected parts of the tuber, the healthy portion was cut almost to the 
edge of the discolored portion and the tuber then broken open, care being 
taken that nothing should touch the advancing margin of the fungus. 
A piece of discolored tissue was taken from the advancing margin and 
put into a tube of melted agar, which was then poured into a Petri dish 
and incubated at room temperature. Six to lo plates were made from 
a single tuber. The plates were watched carefully to be sure that a 
pure culture was the result of the isolation. When this was assured 
a transfer was made to agar slants, and these were kept as stocks. It 
was surprising how very few contaminations appeared in the plates. 
Each culture was numb^ed, and all notes, including source, were kept 
under this number. 
singlb-sporf isoi<ations 
In 1916 when detail work with these cultures was anticipated it seemed 
best to take every precaution to be assured of pure cultures, for § mixed 
culture of two or more species of Fusarium could easily pass unnoticed. 
Therefore, a single-spore isolation was made from eadh culture in the 
following manner: 
A sterile platinum needle was used to transfer spores from the stock 
culture to tube i, which contained 5 cc. of distilled sterile water. From 
this tube a series of five dilutions was made into tubes, each containing 
5 cc. of sterile distilled water. Three 3-mm. loops of material were taken 
from tube i and put into tube 2; three 3-mm. loops of material were taken 
from tube 2 and put into tube 3; and three 3-mm. loops of material were 
taken from tube 3 and put into tube 4. Tubes of standard beef agar were 
melted and poured into Petri dishes and allowed to cool. From each 
dilution tube yi cc. of material was made to flow over the surface of the 
plated agar. Any excess material was drained off. The plates were 
allowed to incubate at room temperature for about 16 hours, when they 
were searched with a microscope for germinating spores. All microscopic 
examination was made through the bottom of the inverted Petri dish. 
Fusarium spores are for the most part hyaline and to locate them on a 
plate of clear agar is very difficult. The scheme was devised of sprinkling 
a few sterile spores of TUletia feotans of wheat over the surface of the agar 
with a tiny sterile spatula. These spores were easy to locate and gave 
the plane of focus in which the Fusarium spores could be found. The 
use of the sterile smut spores proves to be a great timesaver. 
Usually the search for spores began with the more heavily sown plates, 
down through the more dilute ones until a spore was found whi^ was 
sufficiently isolated so that it alone could be removed. The position of 
this spore was marked by a ring of India ink on the glass and it was then 
cut out by means of a stiff platinum cylinder illustrated in Phytopath¬ 
ology (jo) and placed on the upper end of an agar slant. It was carefully 
watched by means of the microscope to be sure that all growth came 
from the spore and not from a piece of mycelium from the edge of the 
piece of agar, as often happened when the germinating spores were 
not sufficiently isolated. Wien it was assured that all growth came 
