﻿BOTANICAL GAZETTE 



The method finally devised and tested is based upon the prin- 

 ciple of dilution, in such a manner as to reduce the possibilities for 

 the occurrence of fungi in the cultures. Poured agar plates were 

 used for this purpose. The method of procedure was as follows : 



Series i.— Plates of 10 different agar media suitable for growing 

 fungi were poured in duplicate. They were potato, oat, cornmeal, 

 rice, bean, raisin, apple, synthetic (Lipman and Brown, N.J. 

 Ann. Rep. 1908. p. 133), soil extract agar (prepared by adding 

 15 gm. agar to 1000 cc. Lohnis soil extract), and Cook's fungi 

 medium no. II (Del. Bull. 91. p. 10). 



After cooling, a block of each medium about 2 cm. square was 

 cut out with a sterile knife, and 1 cc. of sterile soil extract was intro- 

 duced by means of a sterile pipette into the cavity formed. A 

 platinum loopful of a 3 -day old culture of soil organisms in soil 

 extract, known to contain numerous bacteria, protozoa, and fungi, 

 was then carefully rinsed off in the medium. This soil extract 

 was prepared according to Lohnis' directions by heating 1 kg. 

 of good soil with 1 liter of water at 15 lbs. pressure in the autoclave 

 half an hour. It was then removed, mixed with a generous quantity 

 of talc, shaken thoroughly, and the liquid filtered through a double 

 thickness of filter paper, until a clear solution was obtained. The 

 moist residue of soil in the flask was pressed out to remove any 

 solution still remaining. The solution was made up to a volume 

 of 800 cc. and 0.05 per cent K 2 HP0 4 added. 



Series 2. — This served as a check on series 1. consisting of 

 poured plates each inoculated with one loopful of the same 3-day 

 old culture of organisms, and made at the same time, using the 10 

 different media previously mentioned. 



Series 3. — After one week, poured plate cultures were made, 

 using the same media and inoculating with one loopful of the solu- 

 tion taken from the cavities of the agar plates of series 1, in order 

 to make doubly certain that fungi were not present. 



The results in series 1 show that on the plates where a portion 

 of the agar was removed and 1 cc. of soil extract substituted, the 

 bacteria and protozoa developed in enormous numbers, which 

 might be due to the fact that a large surface is exposed for such a 

 relatively small quantity of media. The important point, however, 



