190)).] with especial Reference to its Nitrogenous Metabolism. 463 



operation, their ends were fixed by means of cotton wool in the 

 months of test tubes, and the whole wrapped in filter paper, and left 

 so until actually used. When the samples were removed for analysis, 

 the liquid was at the same time tested for sterility in the way already 

 described. 



The culture liquids employed were sterilised sewage and sewage 

 effluents, and also sterilised artificial liquids whose composition was 

 arranged to resemble that of sewage as nearly as possible. The sewage 

 was usually allowed to stand for several days, then filtered, and after- 

 wards sterilised by successive heatings at 100° C. The artificial culture 

 media were the solution, A, referred to above, and also the same 

 solution to which a small amount of glucose has been added (about 

 0*25 per cent.). 



The methods of analysis were those usually adopted in water and 

 sewage analysis. In the estimation of the free ammonia a small 

 portion of the liquid to be analysed (3 to 10 c.c.) was diluted with about 

 500 c.c. ammonia free water and distilled. Three successive portions 

 of 50 c.c. were distilled off, and the ammonia contained in them esti- 

 mated by means of adding Nessler's solution and comparing the 

 yellow tint with that given by definite amounts of standard ammonium 

 chloride solution. " Albuminoid ammonia " was afterwards estimated 

 by adding a fixed amount of " alkaline permanganate " solution,* 

 distilling as long as ammonia came over in the distillate, and esti- 

 mating these amounts in the same way. It is possible that in some 

 cases the quantities of albuminoid ammonia given in the analyses 

 in the following tables may be slightly too low. For, in certain 

 instances, towards the end of the distillation, the ammonia, came over, 

 in the distillate, very slowly and in very small quantities. It was 

 considered more accurate in these cases to discontinue the analysis 

 when these amounts fell below a certain very small value, since the 

 error in estimating such very small quantities becomes comparatively 

 great. 



The presence of nitrates or nitrites was detected by means of 

 diphenylamine - sulphuric acid and metaphenylene -diamine, while 

 quantitative estimations were made by means of the " copper-zinc 

 couple " method in which the nitrogen present as nitrates or nitrites 

 is estimated as ammonia, f To allow for any ammonia that might 

 have been originally present in the solution or introduced as traces 

 during the analysis, a control estimation was always made. This 

 analysis was carried out in every way like the real one, except that no 

 " couple " was introduced, and the ammonia thus obtained was sub- 

 tracted from that found in the actual estimation. 



The analysis of the cultures of this alga by these methods is by no 



* ' Volumetric Analysis,' Sutton, 8th ed., p. 512. 

 t Ibid., p. 482. 



2 L 2 



