METHODS USED IN THE STUDY OF STARCHES. 



lion* we used >' i v {XT (cut l.ugol'* solution. 



Fur - i i.ii r. .1 tiona were studied . two with raw starch 

 and two with hi tin- first i... the 



.ir. pt.-p.n. -I UK in tin- polarization <.>xaininati"n-. 

 lutinj; solutions of iodine fur the balsam and 

 :iin_ r the -I i. If* in ordinary light with a fully open 

 diaphnipn mill ! p.-w.T. In the I. > '.' <lr<>| 



.'.', j-r i. nt Idol's solution are placed on the 

 . the blidc qmrkly adjusted on the stage of the 

 iiiii-njM.-ope, and the color reaction in quality and quan- 

 tity at once d- I, tlu- quantitative value recorded 

 :i us tin- standard of conipan-..n in relation to 

 other -tar. In -. Here, as in the polarization dctcnnina- 

 it a> found nc.vssary to adopt an arbitrary scale 

 and -MI. h standards. The same scaJe is used an for 

 the polan/atioii \ulues, but the terms light, deep, etc., 

 -11'.-:. in;.-. I for low, high, etc. Moreover, it was 

 found !.. .vssary to modify the selection of starches to be 

 used as standards. The starch of Solatium tuberotum 

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

 that of ( 'rinum moorei as having a value of 50 or " mod- 

 I that of \Vattonia humilu as having a value of 

 :m ..r - light," with corresponding intermediate figures 

 and term* u in the polariscopic determinations. 



The second ex| nnu-nt 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 



The third and fourth experiments are made with 



lioilfd -tar. li with the object of eliciting peculiarities of 



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



\ f ter 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 



nparatvely large amount of the March passes into 



solution, and the remains of the grains appear in the 



form of grain-residues which are made up of partially 



di-integrated grains (capsules with variable amounts of 



content*), 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 '.'" c.c. of water and carefully heated over a bunsen 

 burner only to the point of complete gelatin ization. To 

 f 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 cx|MTinirnt the remainder of the boiled 

 preparation ia boiled for 2 minutes to further break 

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

 I/upnl's solution added ; and then microscopic deter- 

 mination made of the colorations of grain residues, 

 capsules, and solution. 



7. AM LINE 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 parU of indi- 

 vidual grains. Some experimenter* have employed 

 double ..i tuple stains. There is also nu douht that tin- 

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

 at least, many poiuU of much hiatological mij-TUnce, 

 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-Mulct 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 reaction-. 



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 arc 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 n-.i.iion. The 

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

 tions, but the starch standards for these reactions are: 

 Solanum tuberosvm, value 90, " very deep "; Amaryllu 

 belladonna, value 50, " moderate " ; Frrejtia refmrla alba, 

 value 30, "light. " 



8. TEMPERATURES OF GELATIN IZATION. 



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 falla. \ m 

 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 flame, and in the other end was inserted a 

 thermometer which was calibrated in tenths centigrade, 

 but which could readily be read in hundredth*. 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 

 flame. The temperature of the water was raised very 



