VENATION AND SENESCENCE OF POLYEMBRYONIC CITRUS PLANTS 313 
it was necessary to devise a method of clearing and staining so that the 
minutest bundle endings might be readily seen under the microscope. 
An examination of the literature revealed only one or two methods for 
clearing leaves. DeVries (1878), working with potato leaves, gives the 
following directions for clearing: ''Decolorize them in alcohol, treat for a 
longer time with caustic alkali, and finally wash with water and a dilute 
solution of acetic acid. The leaf is then put in glycerine and upon standing 
some time becomes transparent." The method described by Stevens (1907) 
involves, likewise, the use of alcohol for extracting the chlorophyll. This is 
wholly inadequate for the thick, leathery leaves of the Citrus species. 
Therefore a new method was perfected which, with a little modification, 
was found to be practical in clearing all kinds of leaves. 
Entire leaves, if small, or portions of large leaves were placed in a basin 
of water and boiled for some time. This treatment is necessary in order to 
remove water-soluble materials, such as tannin, which prevent an even 
distribution of the stain. 
After the leaves had taken on a water-soaked appearance, they were 
transferred to a side-tube suction flask. This conta'ned 100 cc. of 85 percent 
nitric acid.^ The flask was tightly stoppered, and, by means of a Richard's 
air pump, a partial vacuum was maintained. Such a process accelerated 
the penetration of the acid and at the same time drew off the escaping 
fumes which, if allowed to remain, caused a blistering of the tissue. 
The leaves were allowed to remain in the acid in vacuum from twenty- 
four to thirty-six hours or until they had become transparent. They were 
then washed in running water for six hours, dehydrated, and introduced 
into the stain. After trying a number of stains, e.g., fuchsin, orange G, 
safranin, iodine green, neutral red, and methylene blue, the last was found 
to be by far the most satisfactory. An alcoholic i percent solution was 
found to stain the lignified tissue a deep blue, leaving the remaining parts 
relatively free from stain. The usual time allowed for staining was twenty- 
four hours. Decolorizing in acid alcohol was often necessary. 
Then, following complete dehydration in absolute alcohol and subsequent 
clearing in xylol, the leaves were mounted in castor oil. This medium has 
the following advantages over balsam or glycerine for this work: (i) it 
penetrates more readily than balsam and is easier to handle, and (2) it will 
not take up water as will glycerine. These mounts were made only for 
temporary usage, yet they are still in good condition at the end of two year's 
time. If permanent mounts are desired, balsam may be used to seal the 
edges. 
Modification of the Method for Thin Leaves. The method described 
1 Benedict (unpublished) used concentrated nitric acid for clearing. After allowing 
the leaves to remain in the acid for a while they were removed to a slide and floated in 
glycerine. The slide was then heated over a Bunsen flame until the leaves were trans- 
parent. This method is objectionable because the leaves usually go to pieces. 
