Relation to Factor Limiting Bacterial Activity in Soil. 439 



carried out in a steamer, the temperature of which was regulated by means 

 of a thermostat. Hay-infusion cultures were made after this second treat- 

 ment, and it was then found that no flagellates cropped up. 



The cultures of protozoa used for the inoculation of the bottles of soil 

 were obtained in the following manner : Hay-infusion cultures were made 

 from fresh soil and from old cultures containing cysts which I had on hand. 

 By the use of fine capillary pipettes it was possible to isolate ciliates. which 

 were then sub-cultured in hay-infusion. I found it best to use hay -infusion 

 already containing active bacteria for the sub-culture of isolated forms, the 

 bacteria serving immediately as a source of food for the protozoa. Cultures 

 of flagellates from the 1870 soil were obtained in the same manner, and for 

 the culture of amoebae from the 1870 soil I made use of cysts from pure 

 cultures on agar plates which I had by me. In this way pure cultures of 

 the following protozoa were obtained for use with the 1846 soil : — Col. 

 cucullus, Col. maupasii, Col. steinii, Vort. microstoma, Amenta sp. ?, and 

 Flagellate sp. ?. 



The protozoa for inoculation into the treated Hoosfield soil were obtained 

 by isolation and sub-culture of forms cultivated in hay-infusion from the 

 untreated Hoosfield soil, so that the forms added should represent as nearly 

 as possible the fauna originally present in the soil. The cultures of protozoa 

 thus obtained were one of Amoeba sp. ?, one of Flagellates sp. 1, and one 

 of Ciliates, including Col. cucullus, Col. steinii, and Col. maupasii. The 

 small Ciliate Balantiophorus minutus or elongatus also occurred in 'the 

 cultures made from the untreated soil, but as I was unable to obtain this 

 free from flagellates, the culture of ciliates did not include this form. 



In order to obtain mass cultures of the protozoa in sufficient quantity to 

 serve for inoculation into the soil the following method was employed : 

 80-grm. lots of washed and sterilised sand were put into large sterile petri 

 dishes or glass cylinders and covered with hay-infusion, which was then 

 infected with a pure culture of protozoa, and the latter were allowed to 

 multiply and populate the culture. 



In this way a large quantity of each kind of protozoa was obtained for the 

 inoculation of the soil. This process was carried out by spreading the soil 

 on sheets of sterilised brown paper and then mixing the sand-hay -infusion 

 culture of protozoa into it by means of a sterilised spoon, the whole of the 

 soil, sand, and hay-infusion becoming thoroughly well mixed together and 

 thus ensuring an even distribution of the protozoa throughout the soil. In 

 the case of the 1846 soil the inoculation was carried out in a glass-house 

 which had been steamed down in order to allay dust and thus minimise the 

 chance of infection. The soils were left exposed in this house for some days 



2 N 2 



