438 Mr. T. Goodey. Investigations on Protozoa in 



the limiting factor, presumably the amoebae and flagellates in it, according to 

 Eussell and Hutchinson's hypothesis. 



Besides the above series of samples the set was made up to include a bottle 

 of untreated soil, and one inoculated with a culture of bacteria representative 

 of the bacterial flora added with the cultures of protozoa in the other samples, 

 so as to serve as a check against them ; and a bottle to receive 10 per cent, of 

 the 1870 soil, thus making nine bottles of soil in all. 



Another set of soils was experimented upon at the same time. This 

 consisted of seven bottles of fresh Hoosfield soil, partially sterilised first by 

 toluene and then by heating to 65° C, so as to eliminate the limiting factor, 

 and then inoculated again with cultures of protozoa obtained from the 

 untreated soil. The series consisted of the following: — Untreated, Toluened, 

 Toluened + Untreated, Toluened + Ciliates, Toluened + Amoebae, Toluened + 

 Flagellates, Toluened + Bacteria. The bacteria used for the last-named 

 inoculation were representative of the bacterial flora of the other cultures. 



In each set of bottles the water content of the soil was finally brought to 

 about 18 or 20 per cent, by weight ; this being about the water content at which 

 many of Eussell and Hutchinson's* soils have been maintained, and at which 

 they have found the limiting factor to be active. 



I decided at the outset to make periodic bacterial counts by the gelatine- 

 plate method in order to determine the numbers of bacteria in the soil as 

 nearly as possible once a month and to carry on the experiments for a long 

 period. 



Methods. 



A mixed sample of soil from six bottles of 1846 soil was taken and divided 

 into nine lots of 400 grm. in each. 



Each lot of soil was put up in a quart bottle which had previously been 

 sterilised and plugged with cotton wool. 



In the case of the Hoosfield soil seven 400 grm. lots of slightly air-dried 

 soil were taken after having been passed through a 3-mm. sieve. These 

 were bottled in exactly the same way as the 1846 soil. The soil was first 

 toluened by the addition of 2 per cent, of toluene, which was allowed to 

 remain in the soil for two days, after which the soil was spread out on sheets 

 of paper so as to allow the antiseptic to evaporate. Hay-infusion cultures 

 of the toluened soil were made, and as it was found that flagellates 

 developed in the cultures the bottles of soil were submitted to steam heat at 

 a temperature of 63°-65° C. for three to four hours. This operation was 



* Eussell and Hutchinson, ' Jour. Agric. Science,' vol. 3, Part II (1909), and vol. 5, 

 Part II (1913). 



