METHODS USED IN THE STUDY OF STARCHES. 



27 



what reagents and what concentrations are best adapted 

 for such studies, but the following were finally adopted 

 in this research, although experience has shown that all 

 or nearly all can be modified to advantage in concentra- 

 tion and they can be added to with great profit. Chemi- 

 cally pure chemicals and distilled water were used. The 

 solutions should be made only in small quantities, and 

 when fresh solutions are prepared they must be tested 

 with the several selected starches, the reaction-intensi- 

 ties of which are known, to determine whether or not 

 they are of exactly proper strength. 



Chloral hydrate Schering's chloral hydrate crystals 15 grams, 



water 5 c.c. 



Chromic acid 2.5 grams, water 20 c.c. 

 Pyrogallic acid 4 grama, oxalic acid 0.3 gram, water 35 c.c. 

 Nitric acid 10 c.c. water 35 c.c. 

 Sulphuric acid 10 c.c., water 27 c.c. 

 Hydrochloric acid 9 c.c., water 10 c.c. 

 Potassium hydroxide 0.75 gram, water 55 c.c. 

 Potassium iodide 10 grams, water 30 c.c. 

 Potassium sulphocyanate 5 grams, water 30 c.c. 

 Potassium sulphide 1 gram, water 40 c.c. 

 Sodium hydroxide 0.5 gram, water 100 c.c. 

 Sodium sulphide 1 gram, water 45 c.c. 

 Sodium salicylate 10 grams, water 10 c.c. 

 Calcium nitrate 8 grams, water 16 c.c. 

 Uranium nitrate 8 grams, water 10 c.c. 

 Strontium nitrate 5 grams, water 7 c.c. 

 Cobalt nitrate 9 grams, water 15 c.c. 

 Copper nitrate 15 grams, water 30 c.c. 

 Cupric chloride 9 grams, water 15 c.o. 

 Barium chloride 5 grams, water 12 c.c. 

 Mercuric chloride 18 grams, ammonium chloride 10 grams, 



water 40 c.c. 



Occasionally modified solutions were used in qualita- 

 tive experiments to meet special conditions, note being 

 made in the text at the proper place whenever this has 

 been done. 



In the reactions with the chemical reagents it is 

 essential, in order to obtain uniform and wholly reliable 

 results, that the slides should be prepared with much 

 care as regards the quantity and distribution of the 

 etarch and the quantity of the reagent, and that imme- 

 diately upon the addition of the reagent the preparation 

 be protected so that changes due to alterations in concen- 

 tration and to oxidation will not occur. The method 

 pursued is as follows : 



A square area of starch is first prepared on a slide as 

 in the polarization reactions. This square is surrounded 

 by a layer of purified vaseline 5 mm. wide, applied by 

 an artist's flat camel's hair brush. A cover-slip is now 

 prepared by coating the margin of one surface with a 

 corresponding band of vaseline, so that when the cover- 

 slip is placed on the slide the surfaces of two vaseline 

 squares form an air-tight junction, preventing change in 

 concentration of the reagent by evaporation or absorp- 

 tion of water and eliminating influences of the oxygen 

 of the atmosphere. Two drops of the reagent are care- 

 fully and quickly placed on the center of the starch layer, 

 the cover-slip instantly applied, the slide placed on the 

 stage of the polarizing microscope, a suitable field speed- 

 ily found and examined in polarized light. Usually a 

 practically exact count is made of the number of grains 

 in view, but if the reaction is very rapid this part of the 

 method is modified as hereinafter stated. All these 



procedures are done as expeditiously as possible. In the 

 starches of some species there are to be found variable 

 proportions of very minute grains which for obvious 

 reasons must be ignored in making the count. The num- 

 ber of grains in the field ranges usually from 150 to 200, 

 rarely as few as 75 to 100 or as many as 400 to GOO, the 

 number depending largely upon and in approximate ratio 

 to the mean size of the grains ; but such differences in 

 number do not imply corresponding differences in the 

 total amount of starch present. In specimens in which 

 the grains are small, the number of grains in the field 

 will be larger than when the grains are large, and the 

 number will vary also because of some irregularities in 

 the distribution of the grains, a field always being selected 

 that is well adapted for the count and for watching the 

 processes of gelatinization. Unless gelatiuization occurs 

 very rapidly the percentages of grains and total starch 

 gelatinized are not determined until at the end of 5 

 minutes from the time of the addition of the reagent, 

 and subsequently at 15, 30, 45, and 60 minute intervals, 

 or as may be desirable. At these periods the number 

 of grains not completely gelatinized is counted, and then 

 the percentage of grains completely gelatinized is com- 

 puted by finding the difference between the original 

 number in the field and the number thus found. In 

 addition to the grains completely gelatinized there will 

 be seen grains in partial stages of gelatinization and 

 perhaps some wholly unaffected. The amount of starch 

 remaining ungelatiuized is computed in terms of grains 

 and is estimated by finding the number of grains that 

 are unaffected and the proportions of starch ungelatinized 

 in the partially gelatinized grains. Thus, in the latter 

 case, if there remains an average of one-quarter of 

 the starch unaffected (in some grains it may be one- 

 tenth, in others one-fifth, etc.), it will take 4 grains 

 to represent the amount of starch in an average grain of 

 the specimen, the number thus determined being added 

 to the number of grains that are unaffected, the sum 

 deducted from the original number under observation, 

 computing by the difference the percentage of the total 

 starch gelatinized. 



When gelatinization occurs very rapidly or very 

 slowly the foregoing method must be modified to suit 

 conditions. Frequently complete or almost complete 

 gelatinization occurs within 15 seconds after the appli- 

 cation of the reagent. Obviously time is not permitted 

 for a count of the number of grains in the field before 

 determining the number of grains wholly and partially 

 ungelatinized. By extreme alertness it is possible within 

 15 seconds after the addition of the reagent to have the 

 slide on the stage of the microscope, select a field, make 

 a count of the ungelatinized grains, and estimate the 

 parts of grains that remain ungelatinized. The number 

 of grains in the field can not be satisfactorily counted 

 after gelatinization because of the swollen and distorted 

 condition and overlapping of the grains. Hence, in 

 these very rapid reactions the average number of grains 

 in a field is determined beforehand and a corresponding 

 field is selected. It follows from this that the percentage 

 of starch gelatinized under such conditions is very grossly 

 estimated, that no importance is to be attached to the 



