Compounds in Chloroplasts of Green Cells of Plants. 565 



out to a point were always used instead of steel needles, and also, in order to 

 break up some of the green cells and set free the chloroplasts, a portion of 

 the tissue in each case was still more broken up by turning upon it the blunt 

 end of the glass rod and grinding it between this and the microscope slide on 

 which it was being mounted. 



In choosing tissues for examination, preference so far as possible is given 

 to those where the chloroplasts are more conspicuous in size, and also in some 

 cases, such as Spirogyra, delicate filaments were chosen which could, after 

 extraction as described below with alcohol, be mounted without breaking up. 



In certain cases, such as Plcurococcus, staining can readily be obtained 

 without previous chemical preparation of the tissue, but, in the majority of 

 cases, the lipoids present along with the chlorophyll in the chloroplast prevent 

 the penetration of the stain, also the green colour modifies and masks the blue of 

 the hematoxylin in Macallum's test. For this reason it is well to remove the 

 lipoids and chlorophyll, and in many cases this is by no means an easy task. 

 In some cases standing in cold alcohol removes the chlorophyll quite effectually 

 and leaves the tissue colourless and ready for staining ; but in other cases the 

 tissue may be left at ordinary temperatures for days in alcohol, and this may 

 even be followed by several extractions with ether, and still some of the 

 green colour remains. After a good deal of experimentation the best 

 extractive in these latter cases was found to be boiling alcohol. 



The tissue, either partially teased with the glass points if it is bulky like 

 the leaf of a higher plant or a piece of lichen or moss, or left intact if 

 a delicate structure like an algal filament or Pleurococcm, is placed in water 

 in a watch-glass and then absolute alcohol is gradually added portion-wise 

 and pouring away excess of the mixture at intervals until the fluid is finally 

 all absolute alcohol. The preparation is then boiled in the alcohol and the 

 greenish extract poured away, and this is repeated till the green tissue 

 becomes colourless. The decolorised tissue is then brought back again into 

 distilled water by gradually adding the water to the alcohol, and pouring off. 

 Finally, it is allowed to stand a few minutes in a watch-glass in water 

 redistilled from glass, and is then ready for staining. 



In addition to the unmordaunted simple aqueous solution of well-washed 

 hsematoxylin in |-per-cent. concentration introduced by Macallum* the 

 older histo-chemical tests for iron were also utilised, namely, potassium 

 ferrocyanide and hydrochloric acid for ferric salts, potassium ferricyanide 

 and hydrochloric acid for ferrous salts, and ammonium hydrogen sulphide in 

 glycerine for both. In our opinion the Macallum test surpasses all these in 

 reliability and delicacy. Its only fault is that it is too delicate, and the 

 * Mourn. Physiol.,' vol. 22, p. 92 (1897). 



