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Miss Fraser and Mr. Gardner. Origin and [Apr. 3, 



of pure cholesterol benzoate was obtained, corresponding to 01423 gramme of 

 cholesterol, or - 0745 per cent. This is a higher percentage than we have 

 ever found in healthy animals, even in the case of those fed on diets rich in 

 cholesterol. We have not yet had an opportunity of making further 

 experiments in this direction, but we do not think the result is in disagree- 

 ment with the hypothesis referred to at the beginning of this paper. 



Method of Carrying out the Hwmolytic Experiments. — The method employed 

 is to mix together a suspension of blood corpuscles, a solution of saponin 

 and the serum, making up to a constant volume with physiological salt. 



For this purpose the following solutions are used : — 



(1) Physiological salt — a 0'85-per-cent. solution of NaCl (specially 

 purified) in distilled water. 



(2) Saponin (Merck) — a - 01-per-cent. solution in the physiological salt. 



(3) Eabbit's blood corpuscles — a 5-per-cent. suspension in physiological 

 salt. 



In carrying out the experiments, the mixtures of blood, saponin, serum, and 

 physiological salt are very carefully measured into glass tubes with accurately 

 ground glass stoppers. The tubes are then placed in clamps fitted on to 

 a circular plate of wood in such a manner that they radiate from the centre 

 to the circumference. This disc, with the tubes attached, is slowly revolved 

 in a vertical plane by means of a clockwork drum, the whole apparatus being 

 kept at a constant temperature of 37° by placing it in an incubator. 



As the tubes are completely inverted during the revolution of the disc, the 

 corpuscles are kept equally distributed throughout the mixture without 

 violently agitating the contents. 



Elastic bands hold the stoppers securely in position to prevent their 

 coming out of the tubes as they expand with the heat of incubation. The 

 most favourable period of incubation was found to be three hours. After 

 this the tubes are placed on ice and the corpuscles allowed to settle, or they 

 may be at once centrifugalised. In either case the amount of haemolysis is 

 judged from the colour of the clear supernatant liquid. 



By taking readings of a tintometer [Michael], on consecutive days, it was 

 found that the tint of the tubes kept on ice did not vary, this showing that 

 further haemolysis than that taking place during incubation was prevented 

 entirely by placing on ice. The ice method was adopted generally. 



Experiments to ascertain whether Cholesterol administered with the Food and 

 absorbed by the Animal appears in the Blood Stream as such. 



Experiment I. — It was first necessary to ascertain the least amount of 

 saponin solution which would produce complete haemolysis in a given 



