Relation to Factor Limiting Bacterial Activity in Soil. 441 



A dilution method was first used which Dr. H. B. Hutchinson had devised 

 and which he and I had used in 1910 in the course of some joint work. 

 Ten grm. of soil are shaken up for four minutes with 100 c.c. of 1-per-cent. 

 hay-infusion, either sterile or containing an active growth of soil bacteria. 

 By means of sterile 1 c.c. pipettes varying quantities of this soil suspension 

 are taken out and placed in sterile tubes ; three tubes of each dilution being 

 put up. In the case of the smallest quantities of soil suspension more hay- 

 infusion is added in order to give a sufficient quantity of liquid for purposes 

 of manipulation. The scheme of dilution is as follows : — 



grm. of soil. 



10 c.c. original soil suspension = 1 



5 „ „ =0-5 



2 „ „ = 0-2 



1 „ „ - =0"1 



0-5 „ „ = 0-05 



0-2 „ „ = 0-02 



o-i „ „ =o-oi 



0*5 c.c. mixture of 1 c.c. original + 9 c.c. hay-infusion ... = 0*005 



0-2 „ „ „ „ ... = 0-002 



0-1 „ „ „ „ ... = o-ooi 



The cultures thus obtained are allowed to incubate for about a week and 

 microscopical examination is made of the surface layers by taking out drops 

 on a sterile platinum loop and examining them on glass slides. If protozoa 

 occur in the cultures of any particular dilution, then one infers that they are 

 present in the soil in numbers equal to the factor required to raise the 

 particular dilution to 1 grm. Thus if they occur in all three cultures of 

 0-001 grm., then there are at least 1000 protozoa per gramme of soil. The 

 method possesses the advantage that one deals with a comparatively large 

 quantity of soil, viz. 10 grm., and should thus be able to overcome the 

 difficulties of any irregular distribution of protozoa in the soil itself, provided 

 a good suspension is made. However, in working with it I have obtained 

 most irregular results, which I have not been able to explain, and for 

 this reason I have practically given up using it in favour of an agar-plate 

 method. 



Before leaving the hay-infusion method, however, I may add that I carried 

 out a series of experiments in order to ascertain if the violent agitation of the 

 soil and liquid, in making the suspension by shaking for four minutes, had 

 any injurious effect on living protozoa. I found, when soil was added to 

 a hay-infusion culture containing innumerable active ciliates and apparently 

 no encysting forms and the mixture was shaken violently for four minutes, 

 that on making a series of dilution cultures from this suspension protozoa 



