162 ROYAL SOCIETY OF CANADA 



tageously extracted from tlie bark of the Horse Chestnut (^^sculus 

 hippo-ca,stanum). Between January and March, or January in 

 southern latitudes, is the best time to collect the bark, as the amount 

 of glucoside in the tree is then at its maximum. 



The bark may be stored in a dry condition for an indefinite time, 

 and may be used as wanted. The bark is cut into small pieces and 

 extracted with hot water for a few hours, when the extract will con- 

 tain sesculin, sesculetin and tannin. The two last substances must 

 be removed, as they form black compounds with iron salts; therefore, 

 neutral lead acetate in excess is added to the watery extract, after 

 which evaporate to dryness and extract the residue with hot water, 

 then filter. 



The filtrate will be free from SBsculetin and tannin, but con- 

 tains a considerable amount of lead, which is precipitated with 

 ammonium carbonate, and filtered off. The filtrate is evaporated 

 to a syrup, and the ammonium is thus driven off. The complete 

 removal of the ammonia is indicated by the syrup turning acid. 



The syrup is not chemically pure, but is sufficiently pure for 

 bacteriological work. 



('(iiiji niKifori/ 'rcafii of Black Colonies. 



The following table is an example of the results obtained by 

 testing all the black colonies on aesculin agar plates. Twenty-five 

 colonies were subcultured from four plates made from river water. 

 It will be seen that all were either B. coll or B. afrogenes, excepting 

 numbers 13 and 16. These liquefied gelatine, but gave the usual 

 characteristics of B. co/i. .Similar instances to this are mentioned 

 by Conn and Esten. (5) 



Besides these tests given Ijelow, al)Out 200 black colonies have 

 been tested frora oui- weekly water analyses, all with positive results. 



