192 CHARLES OSCAR CHAMBERS 



MODE OF INOCULATION 



As a check on the amount of nitrogen carried in by the spores 

 it was necessary to adopt some uniform method of inoculation 

 and observe some precaution. The platinum wire was bent 

 at a right angle, making a straight foot 3 to 4 mm. long. The 

 point of this was first moistened by touching it to the solution 

 in the tube to be inoculated, then touched to the mass of spores 

 in the culture tube, taking only what would cling to the point. 

 This was then touched to the surface of the nutrient solution, 

 leaving only the smallest possible number of spores. After 

 each inoculation the remaining spores were all burnt off the 

 wire before the next inoculation. 



A series of determinations on blank inoculations made in 

 this same manner, and also in distilled water, in tap-waters 

 showed that the amount of nitrogen was quite uniform and so 

 small as to be neghgible, less than 0.01 mgm. 



Separate cultures were grown in small test tubes to determine 

 whether any nitrites or nitrates were formed during the growth. 

 These cultures were tested at the end of the period given to the 

 regular cultures and were found not to contain any of the above 

 compounds. The potassium iodide starch test was used for 

 nitrites and the diphenylamine test for nitrates. 

 * Blanks were run to test the effect of a larger or smaller amount 

 of H2SO4 and K2SO4 used in digestion, and there was found to be 

 no apparent difference. Bulletin 31 of the Bureau of Chemistry 

 advises the use of double the usual amount of H2SO4 per K2SO4 

 to prevent foaming. Accordingly, I settled on 2 cc. and 1 gm. 

 K2SO4 for each analysis. No nitrites or nitrates being found no 

 modification of the digestion was thought necessary. 



An unexpected source of error was discovered while testing 

 some blanks supposed to contain no nitrogen, and at the same 

 time some blanks freshly inoculated as described. Both were 

 found to contain the same amount of nitrogen, namely about 

 0.1 mgm. A rigid search on a number of blanks showed that 

 the 1 cc. of H2SO4 used in the digestion was its source. It was 

 found that 1 cc. of the acid alone, put through the entire process 



