MICHIGAN ACADEMY OF SCIENCE. 229 



The spore suspension was made by adding several cubic centimeters 

 of sterile distilled water to an agar slant culture of the organism to be 

 used. The culture was then agitated gently in order to suspend the 

 spores in the water. One cc. of the spore suspension was then intro- 

 duced into a sterile^ cotton plugged test tube by means of a sterile 

 pipette. In this tube had been placed previously 1 cc. of the properly 

 diluted fungicide. This brought the fungicide to the desired dilution. 



The periods for which the spores were subjected to the action of the 

 fungicide were 5, 15^, 30 and 45 minutes. At the end of each of these 

 periods, .2 cc. of the dilution containing the spores was introduced into 

 each of two tubes containing 10 cc. of melted agar cooled to a tempera- 

 ture of 40'^ C. These tubes were shaken thoroughly to disseminate the 

 spores throughout the medium, and their contents at once introduced into 

 sterile petri dishes. A check plate containing spores from the untreated 

 spore suspension was also poured. The plates were allowed to solidify, 

 were labeled and incubated for four days at 28° C. 



It was found by carefully arranging the work that four or five 

 diilerent dilutions could be carried on at one time. Dilution tubes, 

 properly labeled, and in wire test tube racks, greatly facilitated the 

 operation. Each rack was placed immediately in front of the investi- 

 gator. At his right should be placed a copper container with sterile 

 pipettes. The tubes containing melted agar were placed in a water bath 

 regulated to maintain a constant temperature of 40° C. Sterile petri 

 dishes were placed within easy reach and a receptacle for the poured 

 plates placed to the left of the worker. Too much emphasis cannot be 

 placed upon the proper arrangement of all material and apparatus. 

 Upon this depends accuracy and speed in manipulation. Once a series 

 of exposures has been started no time is open for rearranging equipment 

 or replenishing the supply of agar, pipettes, and other necessary material. 



In developing the method it was found that several preliminary tests 

 were necessary in order to determine the approximate dilution strength 

 at which the fungicide would check spore germination and growth. This 

 point varies considerably with different fungicides and with the spores 

 of different organisms. A series starting at a dilution of 1-5 or 1-10 

 and carried through at intervals of 50 up to a final dilution of 1 to 300 

 or in the case of lime sulphur, up to 1 to 700, with a uniform exposure 

 of 15 minutes usually gave the range within which germination was 

 prevented. Table No. 4, in wliich G. rufomaculans was used with 

 Ammoniacal copper carbonate, shows the results from a preliminary 

 test of this kind. 



