BY R. CiREIG-SMITH. 495 



In many cases, the bacteria are seen lying in patches suggest- 

 ing zoogkiea-films, and the bulk of the cells stain faintly with 

 aqueous gentian-violet, a few staining deeply. This is rather 

 characteristic, and, in cases of doubt, aids the positive diagnosis 

 which, however, requires confirmation by growing the bacteria as 

 giant colonies on plates of nutrient agai-, and agar with a plant- 

 extract, e.g.^ beans as a basis. One can generally plant about 24 

 of such colonies upon a single agar-plate, the under-side of which 

 has been divided and numbered with a grease-pencil. In pre- 

 paring the plates, suspensions of the bacteria are obtained by 

 shaking quantities of the soil, varying with the suspected Rhizo- 

 6t?««-content. with -500 c.c. of sterile tap-water for half an hour : 

 from these, dilutions are made. Of each of these dilutions, one- 

 fortieth c.c. is measured in a capillary pipette, and blown upon 

 the agar-plates, smeared, dried at 37° for an hour, inverted, and 

 incubated at 'IT for 5-7 days. 



The preliminary experiments were made with a rich, and a poor 

 Hawkesbury soil, and also a sandy garden (Sydney) soil. In 

 these, the numbers of Rhizobin were 600,000, 24,000, and 3-50,000 

 respectively, in a gram of air-dried soil. The result of the pre- 

 liminary experiment was so satisfactory, that I obtained five soils 

 of known history from the Hawdcesbury Agricultural College 

 through the kindness of the Principal, Mr. H. W. Potts, and 

 employed them in the investigations. In Expt. i., the medium, 

 which originally had an acidity of + 1, was reduced to -I- 0-4, by 

 the addition of three drops of normal soda per 10c. c. In Expt.ii.; 

 the influence of the reaction was tested, largely with the idea of 

 duplicating the tests: and the results showed that the reaction 

 was of importance, and was worthy of investigation. 



EXPERIMKNT i. 



No. I. 



N...2. 

 No. .3. 

 No. 4. 

 No. 5. 



Rhizobi't in 1 grni. of dry soil. 



I, •246.000 



1 23. 000 



J 62,000 







1.51.-) 000 



