96 THE TOXINS OF THE HIGHER PLANTS AND ANIMALS. 



The second, more exact and satisfactory method was introduced pri- 

 marily by Gross and Fuld for presenting the action of trypsin, and was modified 

 by v. Bergmann together with Bamberg and Meyer for the determination 

 of antitrypsin. Numerous workers have found it thoroughly reliable. 

 Its principle is based upon the digestion of a clear casein solution. If the 

 entire amount of casein is digested, no more is left to be precipitated by the 

 addition of acid and therefore the solution remains clear. If, however, 

 casein has been left undigested, the addition of acid will produce a turbid 

 solution or even a white precipitate. 



The necessary reagents are: 



1. Casein Solution. One gm. of casein is dissolved under slight heating in 100 c.c. of 

 N/io Na O H; this solution is next neutralized by N/io HC1, litmus being used as indi- 

 cator, and diluted with physiological salt solution up to 500 c.c. (If sterilized, it can be 

 kept for a long while). 



2. Trypsin Solution. 0.5 gm. of trypsin (purissimum Griibler) is dissolved in 

 50 c.c of NaCL-f 0.05 c.c. of normal sodium hydrate solution and then diluted with 

 physiological saline up to 500 c.c. 



3. Acid Solution. Five c.c. of acetic acid+ 45 c.c. of alcohol + 50 c.c. of water. 



First the titration of the trypsin solution is undertaken in order to find out how much 

 trypsin is required to fully digest a constant quantity of casein. Gradually increasing 

 amounts of trypsin (from o.i to 0.6 c.c.) are placed into six test-tubes and to each 2.0 c.c. 

 of casein are added. These tubes are placed into an ncubator at 37 for one-half hour, 

 and then several drops of the acid solution are placed into each tube. The first tube, and 

 all those above it that remain absolutely clear, contain enough trypsin to fully digest the 

 2.0 c.c. of casein. 



Now comes the second part of the test. 



Into each of eight to ten test-tubes, are placed 2 c.c. of the casein solution and 0.5 

 c.c. of a 2 per cent, dilution of the serum for examination; to these is next added the tryp- 

 sin solution in successively increasing amounts, beginning with the smallest quantity 

 which in the first part of the test was sufficient to completely digest the given amount of 

 casein. Salt solution is then added to each of the test tubes so that all contain an equal 

 quantity of fluid, and the mixtures placed into an incubator at 37 for one-half hour. At 

 the end of this time, several drops of the acid are added to each tube. Those tubes which 

 become cloudy or show a precipitate, designate the amounts of trypsin solution which have 

 been neutralized by the 0.5 c.c. of diluted serum. For example: 



In the first part of the test it was found that the tube containing 0.4 c.c. of trypsin 

 was the first to remain clear, in other words was sufficient to fully digest 2 c.c. of the casein 

 solution. In the second part of the test the lower limit of the added trypsin dilution was 

 0.4, and it was found that the tubes containing 0.4, 0.5, 0.6 and 0.7 c.c. of trypsin, for 

 example, now gave precipitates and only 0.8 remained clear. This indicates that part of 

 the formerly sufficient amount of trypsin was now neutralized by the antitrypsin of the 

 added serum so that digestion was interfered with. Thus the antitrypsin titer in this case 

 is 0.8. 



Recently the above method of trypsin titration has been applied to the determination 

 of the presence of pancreatic ferment in the intestine, feces, and stomach contents. 



