26 



METHODS USED IN THE STUDY OF STARCHES. 



slowly, and the water occasionally stirred, so that at no 

 time did the two thermometers differ more than about 

 2°. As the temperature increased, specimens of the 

 starch were examined at intervals, the tube being shaken, 

 and a specimen obtained by inserting the end of the 

 pipette to the bottom of the tube, a clean pipette being 

 used to remove each specimen. Each specimen was 

 placed on a slide, upon which was recorded both tem- 

 peratures, and the slide was examined in the polarizing 

 microscope. The temperatures at which there is an en- 

 tire loss of anieotropy of a majority and of all of the 

 grains were recorded as the temperatures of the tube. 

 The lower temperature recorded on the slide was the 

 record of the thermometer in the test-tube, and the higher 

 temperature was that of the water-bath. The actual 

 temperature of gelatinization lies somewhere between 

 the two, and for convenience, especially for purposes of 

 comparison, the mean of the two was for obvious reasons 

 taken as the "temperature of gelatinization." In the 

 records all three temperatures are given in accordance 

 with the foregoing. 



9. Action of Swelling Reagents. 



Quite a number of swelling or gelatinizing reagents, 

 of very diverse chemical composition and exhibiting more 

 or less individuality of action, have been used by various 

 experimenters in studies of the structural peculiarities 

 of starch-grains or in the differentiation of different 

 kinds of starch or for other incidental purposes. This 

 method of differentiating starches seemed so promising 

 that in the preceding research five such reagents were 

 selected. For obvious reasons choice was made of those 

 which differ widely in chemical composition and which 

 yield sufficiently prompt and characteristic results. 

 Those selected included chloral hydrate-iodine, chromic 

 acid, pyrogallic acid, ferric chloride, and Purdy's solu- 

 tion. For evident reasons it is desirable to repeat 

 some of the statements made in the preceding memoir. 



The chloral hydrate-iodine solution was prepared by 

 saturating a saturated solution of chloral hydrate with 

 iodine. This solution, sooner or later, not only causes 

 swelling and ultimate partial dissolution of the grains, 

 but, owing to the presence of iodine, also yields important 

 accompanying color reactions ; and it is, on the whole, to 

 be regarded as a very important reagent. 



Chromic acid was used in the form of a 25 per cent 

 solution, and it is the only one of the five reagents that 

 causes, within the periods of observation, a complete 

 disintegration of the grains. It gives rise to gas bubbles 

 during the decomposition processes. 



The pyrogallic-acid solution was prepared by making 

 a saturated solution and diluting this with three parte 

 of water, adding oxalic acid in the proportion of 4 per 

 cent to hinder oxidation. 



The ferric-chloride solution consisted of equal parts 

 of a saturated solution and water. Purdy's solution 

 was made by diluting the standard solution with an equal 

 volume of water. 



The last reagent was usually found to be the least 

 active of the five, and it is, so far as the effects on the 

 grains are concerned, probably essentially an aqueous 

 solution of potassium hydroxide, and therefore likely 

 possesses no advantages, except perhaps in keeping quali- 

 ties, over the simple aqueous solution. Oxygen or ex- 

 posure to the air favors the actions of pyrogallic acid, but 

 hinders those of chloral hydrate and ferric chloride. In 

 the former case, the grains near the edge, or on the out- 

 side, of the cover-slip are decidedly more affected than 

 those within, while with the latter the opposite is true. 



There are some forms of commercial chloral hydrate 

 that have very little action, which may be due to under- 

 hydration or over-hydration. The crystals put up by 

 Schering were used throughout this investigation. 



It is important that fresh solutions of the reagents be 

 prepared at short intervals, as all tend to deteriorate, and 

 it is well to let them stand over night before using. 



In using these reagents a small amount of starch 

 was placed in a slide as in the polarization experiments, 

 several drops of the reagent were added, a cover-glass 

 put on, and the progress of events examined under the 

 microscope. In using a given reagent with a given kind 

 of starch, it was found that there was a certain amount 

 of variation in the effects from time to time, probably 

 attributable to variations in temperature, so that these 

 studies were made as far as possible under constant tem- 

 perature conditions. The variations, as a rule, were 

 unimportant. These agents give rise to gelatinization 

 and swelling of the grain and cause the existence of the 

 outer and inner parts of the grains to appear very con- 

 spicuous — the outer part becoming sac-like and inclosing 

 a less dense or semi-fluid substance. 



Experience taught us that not only the method but 

 also the reagents, as regards both kind and concentration 

 of solution, can be markedly improved. As previously 

 stated, the method though gross seemed to meet the theo- 

 retical requirements of the research — that is, the deter- 

 mination whether or not starches are modified in relation 

 to species and genera — without attempting to establish 

 constants or strictly exact data. During the progress 

 of the present research we used, in a limited number of 

 experiments, certain reagents which in the text that 

 follows are designated: 



SOLUTIOIf No. 2. 

 Chloral hydrate-iodine — Schering's crystals of chloral hydrate 



30 grama, water 17 c.c, Lugol's solution 3 c.c. 

 Chromic acid 10 grams, water 40 c.c. 



Fyrogallic acid 9 grams, oxalic acid 0.5 gram, water 40 c.c. 

 Ferric chloride 50 grams, water 5 c.c. 

 Ammonium nitrate 15 grams, water 10 c.c. 



After a time the ferric chloride was abandoned be- 

 cause of difficulties in standardization and in obtaining 

 satisfactory uniformity in the results of repeated experi- 

 ments, and it was also found that other of the reagents 

 could be used to better advantage in a modified form. 

 A few experiments were also made. with ammonium 

 nitrate and certain other reagents, but for various reasons 

 were set aside. It is yet wholly problematical as to 



