252 PovaH: A CRITICAL STUDY OF 
2. ISOLATION 
As a rule little difficulty was experienced in obtaining a pure 
gross culture, one or two transfers to poured plates usually sufficing 
to eliminate obvious contaminations. Ina series of closely related 
forms, however, there is no certainty of a pure culture, unless the 
latter has originated from a single spore. For practically all 
purposes a medium of the following composition per liter was used: 
SPRANE INO TNS A Vib TERE ea bi F858 ae eh Pe ee op gt I.o gm 
Dihydrogen potassium phosphate................... 0.5 gm 
en LL ESS So APE ie NS ce MND Re: eos NS dca res aa i 0.5 gm 
MA Pierit is ciate eect oie cl een sa tee  t 0.25 gm. 
Came mbar arte ate Sete ee ost 5.0 gm 
PIQUE AG AT Coe Pe ne ae Os ee NT ad I3.0 gm. 
The advantage of this medium is that all forms tried have 
been found to grow on it, and that cultures kept on it through 
the summer months (June-September) retain their vitality, at 
least when kept in a cool place. For stock cultures the writer 
has lately used a formula given him by Blakeslee, viz.: 
PeRRE PAE ee are eh et. en a oie inn ee: 2.0% 
Pepeoee (WHE) OS ce ee ees 0.1% 
RET ORE od oidg ateettcgtminiahis SRO Sk pela ge ort i ts 2.0% 
Dry malt extract (Eimer & Amend)....... Me a sins ets ak sae 
This agar gives a much more luxuriant growth than the first- 
mentioned medium, but its use for keeping cultures longer than 
six weeks has not been tried by the writer. 
Single spore cultures were made by a modified Kauffman’s 
(1908) method, which is essentially an isolation of a single spore 
by spraying a spore dilution on a poured plate. Capillary pipettes 
were made, fitted with a tiny loose-fitting cotton plug immediately 
below the nipple. These were then sterilized in a device made as 
follows: two pieces of corrugated sheet asbestos, two decimeters 
Square, were fastened together with wire in such a way that the 
ridges met so as to form a series of tubes between the two pieces 
of asbestos. The pipettes, minus the rubber nipples, were then 
inserted in these tubes and the apparatus was placed on a tripod 
over a burner and heated. Later, after the apparatus had cooled, 
the rubber nipples were replaced very carefully to avoid displacing 
the sterile air in the pipettes. 
