Example 11 



Preparation of a Transverse Section of a Stem of Aristolochia 

 Stained by the Method of Johansen 



In the preparation of a transverse section of a root described in Example 9, 

 nothing was required except that an outHne of the cells be shown as a skele- 

 ton and that this skeleton be differentiated as to woody and nonwoody tissues. 



The present example describes the preparation of a section of a plant stem 

 preserved, sectioned, and stained in such a manner as to differentiate all of 

 the components present. The technique to be described could be applied equally 

 well to a leaf or any other part of a plant; the stem of an Aristolochia has 

 been selected only because of its wide use in teaching. 



First it is necessary to collect and fix the material. The choice of fixative is 

 not of any great importance, but the majority of workers prefer a "Craf ' fix- 

 ative, such as the formula of Lavdowsky given on page 8, for this type of 

 material. About a pint of this solution should be prepared immediately before 

 it is required for use. 



Great care must be taken in the selection of a stem. If a second- or third- 

 year growth is taken, it will be too woody to cut satisfactorily, while a new 

 growth of early spring will not be differentiated sufficiently to justify applying 

 a complex stain of the type used in this example. The best Aristolochia mate- 

 rial is usually obtained from the new season's growth when it has partially 

 ripened toward the end of August. Care must be taken that the piece of stem 

 is not so cut as to permit the introduction of air into the transporting vessels 

 since this will greatly increase the difficulties of subsequent manipulation. 

 Unless the stem can be cut in the early morning while it is completely turgid, 

 it is best to pull down a branch of the vine and make the first cut under the 

 surface of water in a bucket. About a foot of stem is pushed under the water 

 and cut off. The piece should be transferred immediately to a large volume of 

 fixative and stored in the dark for about three days. At the end of this time 

 the central portion of the stem should be cut into one-half inch lengths with 

 care so as to avoid crushing, and the pieces washed overnight in running water. 



The process of dehydrating, clearing, and embedding plant material is exactly 

 the same as that used for animal material, so that the directions given on the 

 handling of frog intestine in Example 10 may be used without modification. 

 Sections should be cut from the block at about 10 microns thickness. These, 

 if desired, may have the wax dissolved from them directly and be handled as were 

 the sections of root in Example 9- It is much more convenient, however, to 

 mount individual sections or short lengths of ribbon on the slide in the man- 



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