184 CHARLES OSCAR CHAMBERS 



much easier to digest. Hence this kind of control was resorted 

 to from Experiment D to the end. It was even considered 

 useless to analyze these so long as there was no apparent gain 

 in the cultures over that supplied. 



At first, sets of cultures were kept under air-tight bell-jars 

 and aerated by means of a suction pump, drawing the air first 

 through wash bottles, one containing NaOH and the other 

 containing H2SO4 to remove any combined nitrogen. This was 

 later abandoned along with the controls, as already mentioned, 

 and for the same reason. 



It seemed quite significant that when the cultures were ana- 

 lyzed after 30 days that they would yield exactly as much nitro- 

 gen as had been put in at the beginning and no more, as shown 

 in Experiments A, B, and C. This ratio, however, did not hold 

 out, as will be seen by the remaining experiments. 



The water-blank controls kept as above described showed no 

 accumulation of nitrogen when analyzed. The blanks kept 

 with dead spores gave some color when digested and Nessler- 

 ized but later, by the adoption of a special uniform method of 

 inoculation this was reduced to a negligible quantity as detailed 

 on a later page. 



The live spores to which no nitrogen was supplied simply 

 germinated and made a thin ghostlike mycelium but made no 

 real growth and yielded only the same depth of coloration as the 

 dead spores alone yielded. This confirms the statement of 

 Dox that "Nitrogen in some combined form is essential, for the 

 molds {Aspergillus and Penecillium) can not utilize atmos- 

 pheric nitrogen sufficiently to produce a normal colony. Often 

 a slight growth of mold had been observed in the absence of 

 nitrogenous compounds, but this is to be regarded as due rather 

 to the reserve protein present in the spore itself or to a small 

 amount of nitrogenous substance transferred to the medium 

 during inoculation. 



This, according to the experiments and logic of Boussingault, 

 should have been regarded as conclusive evidence against the 

 possible fixation of free nitrogen by Aspergillus niger. However, 

 my results do not agree with a number of investigators, including 

 Puriewitsch and those in the tabulated list. 



