J. BRONFENBRENNER 1059 



from mechanical defects, it is essential to ascertain these qualities in individual thimbles be- 

 fore each test. For this purpose each thimble (Schleicher and Schiill S79-A), whether it be 

 new or used previously, is examined, on the one hand, for its eificiency in holding back the 

 natural protein (20 per cent egg white or normal serum), and, on the other hand, for its per- 

 meability to diffusible protein derivatives (peptone). The whole procedure must be carried 

 out under conditions of strict asepsis, as even a slight amount of bacterial decomposition 

 taking place during the time allowed for the dialysis will vitiate the results. The thimbles 

 are carefully washed and boiled before each test and again immediately after use. Sterile 

 thimbles are stored in sterile distilled water under toluol, in a closed sterile container. 



Ninhydrin test. — The course of the Abderhalden reaction is judged by the appearance of 

 dialyzable protein-split products in the fluid surrounding the thimble. This is ascertained, as 

 stated previously, by a ninhydrin test. The intensity of the color can be used to measure the 

 extent of hydrolysis, provided the concentration of the reagent is strictly uniform in all tests 

 of a given series. For the same reason it is essential to prevent unequal evaporation and con- 

 sequent concentration of the dialysate, and therefore the contents of the dialyzing thimble, 

 as well as the outer fluid, is overlaid with toluol during the incubation. Even traces of im- 

 purity, such as organic particles or free acid or alkali on the glassware, interfere with the test 

 by giving atypical color reactions which may obscure the reading.' Such atypical reactions 

 may, in reality, be due to impurity alone, but at times they may mask a true color test which 

 may be present at the same time. It is therefore necessary to repeat the test in all cases 

 where the color reaction is atypical. 



The test. — The necessity of ascertaining the condition of biological reagents makes it 

 imperative to accompany each test, or series of tests, of unknown serum by proper con- 

 trols. Previous to beginning the test proper, one must ascertain that the substratum has 

 remained free from soluble protein derivatives which by themselves could react with 

 ninhydrin. 



A portion of substratum necessary for the performance of the whole test with its controls 

 is removed from the container by means of sterile forceps, placed in a thoroughly clean and 

 dry test tube, and boiled with four or five volumes of sterile distilled water. After about five 

 minutes of boiling, during which care is taken not to burn the tissue, about 5 cc. of the water 

 in which the tissue was boiled is filtered through a small piece of hardened filter paper into 

 another thoroughly clean test tube. By adding i cc. of i per cent ninhydrin solution and 

 boiling subsequently, one ascertains whether the tissue is satisfactory. Even the slightest 

 trace of violet color appearing on cooling indicates that the tissue must be re-extracted. 



If the tissue is found to be free from soluble ninhydrin-reacting substances, it is washed 

 in sterile distilled water so as to remove any chloroform or toluol which may adhere, and 

 dried between the folds of sterile filter paper. Such tissue (free from an excess of water) is 

 ready for use. In spite of the fact that only substrata free from soluble protein are used for the 

 test, it is still recommended that each be controlled again during the test as follows (Table I, 

 Thimble 7): 



Parallel with the test proper (Table I, Thimble 7) one tested thimble receives about 0.5 

 gm. of the tested substratum. The thimble is held up with sterile forceps and 1.5 cc. of physi- 

 ological salt solution is introduced, sufficient to cover the substratum. Then the open end of 

 the thimble is closed by means of long forceps, while it is washed with sterile distilled water^ 

 to insure the absence of protein on the outside, and placed in an Erlenmeyer flask contain- 



' Daetjen, H., and Fraenkel, "E.: Miinchen. med. Wchnschr., 61, 466. 1914; Abderhalden, E.: 

 Abwekrfennenie (4th ed.), p. 289. Berlin, 1914. 



= All the water used must be freshly distilled and sterilized to avoid possible presence of bacterial 

 protein in it. 



