380 



METHODS AND FORMULAS 



DS 21.10 



acid alcohol, of which two changes of a 

 smaller volume may 1)0 used to remove 

 the last traces of the toluidine bhie. The 

 total time required for differentiation will 

 be from one to two weeks for a specimen 

 of the size indicated, or from three to 

 oijfht months for a ral)l)it embryo two 

 inches long. 



As soon as differentiation is presumed 

 to be complete, the specimen may be de- 

 hydrated, cleared, and mounted. Nothing 

 is necessary to render these specimens 

 permanent other than the maintenance of 

 an acid environment, and it is therefore 

 recommended that 0.1% of hydrochloric 

 acid be added to all the alcohols used Jor 

 dehydration. The present specimen, which 

 can be prepared as a microscope slide, 

 may then be dehydrated in xylene and 

 mounted in any acid mounting medium, 

 such as an old batch of Canada balsam 

 or one of the synthetic resins to which 

 salicylic acid has been added. 



Preparation of cleared museum speci- 

 mens is a great deal more difficult. The 

 mounting medium employed is usually 

 benzyl benzoate to which has been added 

 1 % of methyl sahcylate. The objection to 

 this medium is its sensitivity to water; 

 e\en a small specimen will require de- 

 hydration in many changes of absolute 

 alcohol before it can be mounted. 



Preparation of a transverse 



section of a root using the acid 



fuchsin-iodine green technique 



of Chamberlain 1915 



This is the simplest of all the prepa- 

 rations described in the present section 

 of the work, and can be unhesitatingly 

 recommended to the beginner who has 

 never previously prepared a section of any 

 type. This preparation is designed only to 

 show the skeletal outlines of the cells, the 

 cytological contents of which are removed 

 in the course of the preparation. If cyto- 

 logical detail in a botanical section speci- 

 men is required, reference should be made 

 to the typical preparation of a plant stem 

 described in the last chapter. 



It does not matter from what source 

 the root is obtained, but the beginner 

 should select some soft root of about 



>s inch, or rather less, in diameter. If the 

 root is collected from a living plant, it 

 should be thoroughly washed to remove 

 any adherent sand grains, which would 

 spoil the edge of the cutting knife, and 

 then preserved in 95% alcohol until lo- 

 ([uircd. The 95% alcohol should be 

 changed as it becomes discolored, but 

 with this precaution the specimens may 

 be preserved indefinitely. 



It is even possible to make preparations 

 of this type from dried roots which have 

 been preserved in a herbarium. The best 

 method of swelling and softening these 

 dried preparations is that recommended 

 byLangeron (Langeron 1942, 1263) which 

 requires a 10% solution of phenol in lactic 

 acid. The lactic acid employed is the 

 ordinary commercial solution, in which 

 the phenol should be dissolved immedi- 

 ately before it is required. Pieces of the 

 dried root are then placed in a reasonably 

 large volume of this material and heated 

 over a low flame to a temperature of about 

 50°C. Within 10 or 15 minutes a com- 

 pletely dried herbarium specimen will 

 have become swollen out to its normal 

 size and softened to the extent that sec- 

 tions may readily be cut from it. 



The method of sectioning does not par- 

 ticularly matter, but since the sections 

 cannot in any case be subjected to the 

 first process while they are attached to the 

 slide, there is no real advantage in em- 

 bedding in paraffin and cutting in this 

 medium if an ordinary hand microtome is 

 available. Sections can be taken from this 

 microtome by (see Chapter 9) holding 

 them either in pieces of pith or between 

 the cut halves of a carrot. If the sections 

 are cut by hand, they may be transferred 

 immediately after they are cut to a dish 

 of 20% alcohol, and from there to water; 

 if they are cut in paraffin they should be at 

 least 20 microns in tliickness, and the 

 ribbon, as it is removed from the micro- 

 tome, should be dropped directly into a 

 watch glass of xylene in which the paraf- 

 fin will dissolve. The individual sections 

 are then removed from the xylene with a 

 section lifter, passed through absolute al- 

 cohol for the removal of the xylene, and 

 thence downgraded through alcohols until 

 they reach water. By whatever method 



