434 Bulletin 315 



removed from the tube again for some distance. This method of pro- 

 cedure was followed until ten dishes had been inoculated, when the soil 

 had been quite generally sampled. After inoculation the dishes were set 

 aside at room temperature and the fungi allowed to develop. 



Bacteria usually grow well for a short distance about the planting, 

 but in the course of a few days the fungi very readily outgrow them and 

 transfers to other plates can be made. Pure cultures were often obtained 

 in this way, but for absolute certainty dilution plates were always Doured 

 after the fungi had fruited in the transfer plates. 



In passing it should be stated that whenever isolations were being made, 

 uncovered plates were usually allowed to stand exposed in the culture 

 room in order to determine the possible contaminations arising in the 

 inoculated plates. A fungus was never accepted as being isolated from 

 the soil unless it showed up repeatedly in the plates of the same isolation. 



The tube containing sample C was sterilized externally, as already 

 indicated for the above-mentioned cultures. The soil was transferred 

 from the tube to sterile wide-mouthed bottles by means of a sterile spatula. 

 The soil was mixed as thoroughly as possible by stirring it with a sterile 

 glass rod. Approximately 2 grams was transferred to 100 cc. of sterile 

 water in an Erlenmeyer flask. After a thorough shaking, i cc. was trans- 

 ferred to 9 cc. of sterile water contained in a test tube. From these 10 

 cc. a transfer of i cc. was made to 9 cc. of water. The resulting 10 cc. 

 was used for pouring ten plates, i cc. to a plate, thus making a dilution 

 of 1-5,000. 



Ihe soil of samples F, G, and H was brought to the laboratory and 

 allowed to stand in the bottles over a radiator for four hours. In order 

 to obtain an average of the sample it was now placed in a sterile tin soil 

 sieve, such as is used in soil technology. This instrument makes an ideal 

 apparatus for this purpose, as it is provided with a tin cover and receiver 

 that fit snugly over the top and bottom of the sieve. The only possible 

 contamination arises, therefore, in the transfer of the soil. The sieve 

 was always sterilized by passing it through a flame a number of times 

 just prior to using. In sifting the soil, the lid was occasionally partly 

 removed so that a spatula could be introduced to crush some of the larger 

 particles of soil. Considerable difficulty arose in sifting the soil sample 

 taken from the uppermost part of the soil, because of its wetness. 



After sifting had been completed, about one gram (dry weight) of soil 

 was taken from the receiver and weighed out in a tared, wrapped, and 

 sterilized watch glass. The soil was now transferred to 100 cc. of sterile 

 water in an Erlenmeyer flask and thoroughly shaken. As quickly as 

 possible, and during some agitation of the flask, i cc. was taken and then 

 transferred to 9 cc. of sterile water in a test tube. This 10 cc. (dilution 



