148 ROYAL SOCIETY OF CANADA 



By following these directions exactly satisfactory and even results 

 will be obtained. We have to emphasise here the different manner 

 of neutralisation from that recommended for ordinary media by the 

 Committee on Standard Methods of the American Public Health 

 Association, as the procedure outlined above is absolutely necessary. 

 The main point of our reaction is the forming of the black coloured 

 salt in sufficient quantity to form an easily visible field. This leads 

 us to consider the properties of salt solution as they are explained 

 by physical chemistry. The colon bacillus acting on œscuUn in 

 the absence of other sugars breaks it down to glucose and sesculetin. 

 The glucose is assimilated and the sesculetin gives with iron (ferri) 

 salts a black discoloration. To obtain a reaction with sesculetin 

 the iron salt must be dissociated, as it is the iron ions which produce 

 the combination. We need as many iron ions as are necessary 

 to convert a sufficient quantity of sesculetin into the black salt 

 to form a plainly visible black field. In liquid a^sculin media the 

 concentration of the iron ions is of no importance, as at the moment 

 the state of equilibrium is destroyed, currents in the liquid supply 

 more ions, and we obtain a diffuse discoloration. 



Although agar plates contain only 1.5 per cent of solid matter 

 currents are impossible, and all changes must take place by diffu- 

 sion. As soon as all the iron ions are combined in the immediate 

 vicinity of the colony, the black salt cannot increase in amount 

 until new iron ions are brought to that part by diffusion. This 

 diffusion takes some time, and in the meanwhile the sesculetin, 

 which is also subject to diffusion, has spread over a large area, is in 

 consequence diluted, and thus causes a field of larger dimensions 

 but of less prominent colour (brown-Isabellinus of Saccardo). (2.) 

 Our practical experience amply confirms these statements. 



In the directions for the preparation of sesculin media it has 

 been stated that an acidity of 4~ 0-6 obtained by the addition of 

 iron citrate gave the best results, and it is now necessary to outline 

 and explain the theoretical considerations. 



Iron citrate is a combination of a strong acid and a weak base, 

 and following van't Hoff's law, hydrolysis will take place. Thus : — 



Ca B, (COO), Fe = Fe + C3 H^ (C00)3 



Citrate of iron = Iron + citrate 

 Fe (ions) + H + H = Fe (0 H)3 + H 



This change will. continue until there is an equilibrium between 

 the iron ions and the citric acid. 



