METHODS USED IN THE STUDY OF STARCHES. 



25 



tions we used 0.125, 0.25, and 2 per cent Lugol's solution. 

 Four serial reactions were studied ; two with raw starch 

 and two with gelatinized starch. In the first two, the 

 slides are prepared as in the polarization examinations, 

 substituting solutions of iodine for the balsam and 

 examining the slides in ordinary light with a fully open 

 diaphragm and low power. In the first reaction 2 drops 

 of 0.25 per cent Lugol's solution are placed on the 

 starch, the slide quickly adjusted on the stage of the 

 microscope, and the color reaction in quality and quan- 

 tity at once determined, the quantitative value recorded 

 being taken as the standard of comparison in relation to 

 other starches. Here, as in the polarization determina- 

 tions, it was found necessary to adopt an arbitrary scale 

 and starch standards. The same scale is used as for 

 the polarization values, but the terms light, deep, etc., 

 were substituted for low, high, etc. Moreover, it was 

 found necessary to modify the selection of starches to be 

 used as standards. The starch of Solarium tuberosum 

 was taken as having a value of 60 or " moderately deep," 

 that of Crinum moorei as having a value of 50 or " mod- 

 erate," and that of Waisowia humilis as having a value of 

 30 or " light," with corresponding intermediate figures 

 and terms as in the polariscopic determinations. 



The second experiment is made, using 0.125 per cent 

 solution, often bringing out color peculiarities which may 

 be obscured or not be observed when the reagent is 

 stronger. 



The third and fourth experiments are made with 

 boiled starch with the object of eliciting peculiarities of 

 reaction of the grains, solution, grain-residues, and cap- 

 sules. After heating the grains until complete gelatiniza- 

 tion occurs a variable amount of the starch passes into 

 solution, so that both grains and solution give starch 

 reactions. Upon boiling the preparation for 2 minutes 

 a comparatvely large amount of the starch passes into 

 solution, and the remains of the grains appear in the 

 form of grain-residues which are made up of partially 

 disintegrated grains (capsules with variable amounts of 

 contents), together with some capsules that are almost 

 or wholly free of starch contents. 



In the third experiment 0.05 gram of starch is placed 

 in 30 c.c. of water and carefully heated over a bunsen 

 burner only to the point of complete gelatinization. To 

 2 c.c. of this preparation is added 2 c.c. of a 2 per cent 

 Lugol's solution, and then the colorations of grains and 

 solution are determined by microscopic examination. 



In the fourth experiment the remainder of the boiled 

 preparation is boiled for 2 minutes to further break 

 down the starch grains; then 4 c.c. of the 2 per cent 

 Lugol's solution added; and then microscopic deter- 

 mination made of the colorations of grain residues, 

 capsules, and solution. 



7. Aniline Reactions. 



A number of anilines have been found by various 

 investigators to be of value in the differentiation of 

 starches from different sources, of different grains of 



the same kind of starch, and of different parts of indi- 

 vidual grains. Some experimenters have employed 

 double or triple stains. There is also no doubt that the 

 use of double or triple stains would bring out, at times 

 at least, many points of much histological importance, 

 but this would have involved the carrying out of the 

 histological examinations in such detail as to be pro- 

 hibitive in a research of this character. Safranin and 

 gentian-violet were selected, not because they are prob- 

 ably the best of these stains for differential purposes, but 

 because they have been found very useful in starch exam- 

 inations and as they yield single color reactions. 



Aniline colors in solution, especially when in weak 

 solution and exposed to light, are notably unstable, and 

 in order to secure strictly comparable results a quantity 

 of a relatively strong standard solution was prepared 

 and kept in the dark, tightly corked. The stock solutions 

 were composed of 0.25 gram of aniline with 150 c.c. of 

 distilled water. From day to day dilute solutions were 

 prepared by adding 33 c.c. of water to 2 c.c. of the stock 

 solution; 15 c.c. of the latter solution are placed in a 

 test-tube containing 0.07 gram of starch, the preparation 

 agitated, 1 or 2 drops withdrawn in a minute and exam- 

 ined under the microscope, and a final examination made 

 at the end of half an hour. In these color determina- 

 tions the microscope is used, as in the iodine reactions, 

 with a fully open diaphragm and low power. Owing to 

 the relatively slow reaction, the values for comparative 

 purposes were taken at the end of a half hour instead 

 of immediately, as in the first iodine reaction. The 

 method of valuation is the same as in the iodine reac- 

 tions, but the starch standards for these reactions are : 

 Solanum tuberosum, value 90, " very deep " ; Amaryllis 

 belladonna, value 50, " moderate "; Freesia refracta alba, 

 value 30, " light." 



8. Tempeeatuees of Gelatinization. 



While the records of various investigators indicate 

 that there are more or less marked differences in the 

 temperatures of gelatinization of different kinds of 

 starches, and even in case of different grains of the 

 same starches, the figures applying to the same kind 

 of starch are generally so at variance that not much value 

 is to be attached to them. The sources of fallacy in 

 such observations, unless the determinations are made 

 with the greatest precautions, are well known to every 

 biochemist. We therefore carried out this work with 

 especial care. A long quadrangular water-bath was 

 used, holding about 4 liters of water ; one end was placed 

 over the gas fiame, and in the other end was inserted a 

 thermometer which was calibrated in tenths centigrade, 

 but which could readily be read in hundredths. A small 

 quantity of starch with 10 c.c. of water was placed in a 

 test-tube, into which was inserted, through a perforated 

 cork, a thermometer similar to the one in the water- 

 bath, and the test-tube immersed in a suspended wire 

 basket in the part of the water-bath farthest from the 

 fiame. The temperature of the water was raised very 



