424 Bulletin 315 



(indication of decomposition of organic matter, hence complete sterilization). In like 

 manner all the glassware (test tubes, pipettes, flasks, etc.) was sterilized. 



At the time of collecting the samples, after removing the leaf mould layer, the 

 cotton plug was removed from the test tube which was immediately inserted vertically 

 in the ground with a gentle rotary motion to a depth of about 3 to 5 cm. This test 

 tube was withdrawn and the cotton plug immediately replaced. In this manner the 

 uppermost part of the soil, which contains the greatest number of bacteria, was obtained. 

 In cases where it was desirable to collect the samples from the deeper portions of the 

 soil, a vertical cut was made and, after scraping the surface with a sterilized spatula, 

 the sample was collected as above indicated, except that the test tube was inserted 

 horizontally at the desired depth 10 to 12 cm. below the surface. 



For the study of the moulds, the samples were taken from that part of the leaf 

 mould layer next to the soil after the t6p leaves were carefully removed. The material 

 was collected in sterile test tubes and inoculated on nutrient agar plates by touching 

 and gently rubbing the material on the surface of the medium. The plates were incu- 

 bated at 37° C. and at room temperature for some days, after which a luxuriant growth 

 was obtained. This collection was made during autumn, when the growth of moulds 

 on the ground was found to be more luxuriant. 



Beckwith ('11), in North Dakota, found representatives of the following 

 genera to be present in what he terms " wheat-sick soil" : Fusarium, two 

 species; Colletotrichum, two species; Macrosporium, Alternaria, Spicaria, 

 Verticillium, Rhopalomyces, Cephalothecium, and Helminthosporium. 

 It would be highly interesting to know whether the same results would 

 follow provided isolations were made in late summer, autumn, and winter, 

 presuming, however, that the above results followed isolations made in 

 May, June, and July. While some of the above fungi have been found 

 repeatedly in the soil, this is the first report of the isolation of others. 

 Without questioning the results, it seems peculiar, however, that no 

 Penicillia nor Mucors should have been encountered, as species of these two 

 genera have been quite generally isolated from the earliest investigations 

 to the present. 



In describing his method of isolation and the kind of media used, citation 

 is made from the original : 



A soil solution was made by mixing one part of soil with ten parts of distilled 

 water. This mixture was agitated frequently for twenty-four hours, in order that such 

 readily soluble matter as was present in the soil might go into solution. This solution 

 was then filtered through Pasteur bougies in order to clarify it, and then, without 

 further treatment, was made into a solid medium by the addition of one and one half 

 per cent of agar-agar. The reaction of this medium was very slightly acid, being .02 

 per cent normal. 



Soil samples were collected from a depth of two inches below the surface, using 

 proper precautions to guard against contamination, and a suspension made by adding 

 one gram of the sample to 10 cc. of sterile distilled water. After two minutes agitation 

 in order to obtain as even a mixture of the soil particles and water as possible, this sus- 

 pension was plated out on the soil agar. Plates were incubated four days at 29°-30° C. 

 After growth had taken place, the cultures were purified. 



We may well close this historical discussion by taking into consideration 

 the second method mentioned in the introduction for the determination 



('11) Beckwith, T. D. Root and Culm Infections of Wheat by Soil Fungi in North 

 Dakota. Phytopathology i: 170-176. 191 1. 



