Apr. JO, 1916 Citrus Canker 89 



This method consists essentially in the separation of the material into 

 three fractions. Fractions i and 2 consist of the soluble portion ex- 

 tracted by the action of alcohol, ether, and water, and fraction 3 con- 

 sists of the insoluble residue. Fractions i and 2 are separated by lipoid 

 precipitation. The former fraction contains precipitated lipoids, while 

 the latter contains all nonlipoid materials, soluble in alcohol, ether, and 

 water. Instead of the modified Wiley extraction apparatus employed 

 by Koch and his pupils, a rubber analysis extraction apparatus (8) has 

 been employed. Extractions in this apparatus, like those with the 

 modified Wiley apparatus, are carried out at the boiling point of the 

 solvent. 



In making the first alcohol extraction the preserved material was 

 transferred to Schleicher and Schiill extraction thimbles, previously 

 fitted into the siphon cups of the extraction apparatus. The preserving 

 liquid was then filtered through these thimbles. Perforated porcelain 

 plates or filter paper cut to fit were then used as covers over the material 

 in the thimbles. 



Extraction for 12 hours with redistilled 95 per cent alcohol followed. 

 The alcohol was changed two or three times during this extraction in 

 order to prevent possible decomposition of extracted materials in the 

 boiling alcohol. The tissue was pressed to remove the excess alcohol, 

 and an ether extraction was made. This extraction was continued for 

 1 2 hours for the purpose of facilitating the subsequent powdering of the 

 tissues. The material was then removed from the extraction thimbles 

 to a mortar and was ground to a powder. This powder was placed in a 

 stoppered flask with a volume of distilled water equaling twice the fresh 

 weight of the material and was boiled on a steam bath for two hours. 

 Warm absolute alcohol was added in a sufficient quantity to bring the 

 alcohol content of the whole up to 90 per cent. The mixture was warmed 

 on the bath, with repeated shaking, and set aside until the following day. 

 It was then filtered through the original extraction thimbles and extracted 

 for 12 hours with 95 per cent alcohol. At the close of this extraction 

 the residue in the cups (fraction 3) was transferred to previously weighed 

 porcelain crucibles and dried to constant weight in an oven at 100° C. 

 By this procedure the alcohol, ether, and water-soluble portions (frac- 

 tions I and 2) were separated from the insoluble portion (fraction 3). 



In further preparing the soluble portions of the material for analyses 

 they were combined only after the ether-soluble portion had been heated 

 on a water bath until the odor of ether could no longer be detected. A 

 little alcohol was added from time to time to take up the materials left 

 behind by the loss of ether by evaporation. In pouring the solutions 

 together a precipitate appeared which was rendered soluble by the addi- 

 tion of sufficient hot water to bring the alcohol concentration down to 70 

 per cent. The solution was then made up to 2,000 c. c, 200 c. c. of 

 which were taken for the estimation of solids. The remainder was 



