CHAPTER II. 



METHODS USED IN THE STUDY OF STARCHES. 



The methods used in the preceding research (Publi- 

 cation No. 173) were at its inception sufficiently satis- 

 factory to meet the theoretical requirements of a purely 

 preliminary and exploratory investigation, but as the 

 work progressed it was found, as was to be expected, 

 that radical improvements could be made in various 

 directions. Advantage has been taken of this experience, 

 and while the methods continue to be inexact, in the 

 conventional sense, they are practically exact so far as 

 satisfactory differentiation and recognition of different 

 starches are concerned. For obvious reasons the descrip- 

 tions of the methods given in the previous research are 

 herein in a large measure repeated, with some omissions, 

 modifications, and additions. 



1. PREPARATION OF THE STARCHES. 



The starches were prepared from bulbs, tubers, rhi- 

 zomes, bulbils, and pseudobulbs, all in the resting state. 

 The specimens were comminuted by the aid of an ordi- 

 nary culinary grater. Four or five volumes of water are 

 added to the pulp, the mass strained through four thick- 

 nesses of cheese-cloth, and the pulp then washed with 

 sufficient water and strained as before. The starch-water 

 preparation is decanted in cylinders and the starch is 

 cleansed by repeated washing and decantation. Finally 

 the starch is collected in shallow dishes, the water as far 

 as possible drained off, and the preparation dried at 

 a temperature of 50 C. By this simple means starches 

 can be prepared which are with rare exceptions practi- 

 cally free from gross impurities. To have carried out 

 purification to the extent of practical demiueralization 

 would have proven of far greater disadvantage than gain. 



2. SIMULTANEOUS STUDIES OF STARCHES OF THE 

 PARENTS AND HYBRID AND OF THE MEMBERS 

 OF A GENUS. 



For obvious reasons, in a comparative investigation 

 such as the present it ist desirable to make simultaneous 

 examinations of all three or four starches of a set by 

 one of the various methods of study and to take up the 

 methods seriatim in preference to taking one starch 

 and subjecting it to the entire series of methods before 

 studying another specimen; the same plan commends 

 itself when there is a number of sets belonging to the 

 same genus. 



3. HISTOLOGIC METHOD. 



This method has been found to be of signal useful- 

 ness, and up to recent years it has been the sole reliance 

 in attempts to determine the kind of starch. It was, 

 however, perfectly obvious at the very inception of these 

 researches, and rendered clear as far back as the investi- 



gation of C. Nageli in 1858, that this method, unless 

 associated with others, could not be depended upon, and 

 that it was liable to be absolutely misleading. Moreover, 

 differences in form may not in the least imply differences 

 in the starch-substance, as has been pointed out in early 

 chapters of the preceding memoir. Magnification rang- 

 ing from 85 to 400, sometimes higher, was used, accord- 

 ing to the size of the grains and incidental conditions. 

 A sufficient amount of dried starch was disseminated on a 

 slide and mounted in a very dilute Lugol's solution, care 

 being taken not to add a larger quantity of iodine than 

 is sufficient to accentuate the lamella. Since starches 

 of different sources show wide differences in the intensity 

 with which they become colored with iodine, it was found 

 convenient to have on hand a number of solutions rang- 

 ing from 1 to 2 per cent down. By the aid of such ordi- 

 nary microscopic technique there were recorded the 

 form and size of the grain ; the position and form of the 

 hilum; the form, number, and other characteristics of 

 the lamellae; the characteristics pertaining to the form 

 of the grains, whether single or in doublets, triplets, 

 aggregates, etc. In describing the grains the terms 

 " proximal end " and " distal end " have been adopted, 

 the former being the end nearer which the hilum is 

 located. The " longitudinal axis " corresponds with an 

 imaginary line, extending from the proximal end through 

 the hilum to the distal end. In different starches and 

 in different grains of the same kind of starch this may 

 be the long or the short axis. The measurements of 

 eccentricity of the hilum have reference to the distance of 

 the hilum from the proximal end of the longitudinal axis. 



4. PHOTOMICEOORAPHIC KECOKDS. 



Verbal descriptions of the histological characteristics 

 of starch-grains fail to convey adequate conceptions. 

 The notes included in the text have therefore been accom- 

 panied by photomicrographs of the grains lightly colored 

 with iodine, as seen in the microscope. In making these 

 photographs we used an ordinary Bausch and Lomb 

 microscope with a ^-inch objective and a 2-inch eye- 

 piece, which gave us a magnification on the field of 

 projection of 300 diameters. For obvious reasons, many 

 of the more minute features' of the grains will not be 

 seen in the photomicrographs. Moreover, inasmuch as no 

 two fields are alike in case of any starch or slide, the 

 pictures are to be taken as being grossly of an average 

 character of a field. In recording the histological de- 

 scriptions, especially as regards variations in form, many 

 fields were examined. 



The photomicrographs of the plant tissues were 

 made by the use of a l^-inch objective and a 2-inch 

 eye-piece (draw-tube in), or a %-inch objective and a 



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