266 PHYSIOLOGICAL CHEMISTRY 



12. Ortho-tolidin Test (Ruttan and Hardisty). 1 To i c.c. of a 4 per cent 

 glacial acetic acid solution of o-tolidin^in a test-tube add i c.c. of the solution 

 under examination and i c.c. of 3 per cent hydrogen peroxide. In the presence 

 of blood a bluish color develops (sometimes rather slowly) and persists for some 

 time (several hours in some instances). 



This test is said to be as sensitive for the detection of occult blood 

 in feces and stomach contents as is the benzidine reaction. It is also 

 claimed to be more satisfactory for urine than any other blood test. 

 The acetic acid solution may be kept for one month with no reduction 

 in delicacy. 



13. Benzidine Reaction. This is one of the most delicate of the 

 reactions for the detection of blood. Different benzidine prepara- 

 tions vary greatly in their sensitiveness, however. Inasmuch as ben- 

 zidine solutions change readily upon contact with light it is essential 

 that they be kept in a dark place. 



The test is performed as follows: To a saturated solution of benzidine in 

 alcohol or glacial acetic acid add an equal volume of 3 per cent hydrogen peroxide 

 and i c.c. of the solution under examination. If the mixture is not already acid 

 render it so with acetic acid, and note the appearance of a green or blue color. 

 A control test should be made substituting water for the solution under 

 examination. 



The hemoglobin decomposes the hydrogen peroxide (catalysis) and 

 the liberated oxygen oxidizes the benzidine. The sensitiveness of the 

 benzidine reaction is greater when applied to aqueous solutions than 

 when applied to the urine. According to Ascarelli 3 the benzidine reac- 

 tion serves to detect blood when present in a dilution of 1:3,000,000. 

 Walter 4 has also shown the test to be very delicate, and claims it to be 

 more satisfactory than the guaiac test. 



Lyle, Curtman and Marshall 5 have investigated the benzidine 

 reaction very carefully. They suggest a new procedure in preparing the 

 reagent and in conducting the test. 



The test follows: Into a perfectly clean dry test-tube introduce 1.4 c.c. 

 benzidine solution, 6 add 0.2 c.c. of water or glacial acetic acid, then i c.c. of the 



1 Ruttan and Hardisty: Canadian Medical Ass' n Journal, Nov., 1912; also Biochemical 

 Bull., 2, 225, 1913. 



2 NH 2 NH 7 



CeH4 CeEU. 



CH, CH 8 



3 Ascarelli: // policlin sez. prat., 19.09. 



4 Walter: Deut. med. Woch., 36, p. 309. 



6 Lyle, Curtman and Marshall: Jour. Biol. Chem., 19, 445, 1914. 



6 Benzidine solution may be prepared as follows: Place 4.33 c.c. of glacial acetic acid in 

 a small Erlenmeyer flask, warm to 50 and add 0.5 gram of benzidine. Heat the flask for 

 eight to ten minutes in water at 50. To the resultant solution add 19 c.c. of distilled 

 water. This solution may be kept for several days without deterioration. % - 



