262 PHYSIOLOGICAL CHEMISTRY 



9. Hemagglutination. The common garden bean, such as the 

 Scarlet Runner, 1 contains a protein substance which exhibits the 

 interesting property of causing a clumping or agglutination of red 

 blood corpuscles. 2 



Dilute defibrinated blood 3 ten times with physiological sodium chloride solu- 

 tion (0.9 per cent) and place i c.c. in each of three small test-tubes. 



Grind three beans in a coffee mill, or with mortar and pestle to a fine meal 

 and extract for a few minutes with 0.9 per cent sodium chloride solution. Filter 

 and add 0.05 c.c. (about 2-3 drops) of the filtered extract to the first of the blood 

 tubes ; o.oi c.c. to the second ; and 0.05 of 0.9 per cent sodium chloride to the third. 



Invert each tube to mix the contents thoroughly, and note the rapid agglutina- 

 tion and precipitation of the blood corpuscles in the first tube, a less rapid aggluti- 

 nation in the second, while the third or control tube remains unaltered. In 

 one -half hour the corpuscles in the first tube often are packed solid and one is 

 able to pour off perfectly clear serum. 



If the remainder of the bean extract is boiled for a few minutes, the coagulum 

 filtered out and 0.05 c.c. of the filtrate added to the control tube, still no agglutina- 

 tion occurs, indicating that the hemagglutinin has been destroyed or removed 

 by the boiling. 



10. Diffusion of Hemoglobin. Prepare some hemolyzed ("laky") blood, 

 thus liberating the hemoglobin from the erythrocytes. Test the diffusion of the 

 hemoglobin by preparing a dialyzer like one of the models shown in Fig. 2, page 

 24. How does hemoglobin differ from other well-known crystallizable bodies? 



11. Guaiac Test. To 5 c.c. of water in a test-tube add 2 drops of blood. 

 By means of a pipette drop an alcoholic solution of guaiac (strength about i : 6o) 4 

 into the resulting mixture until a turbidity is observed and add old turpentine 

 or hydrogen peroxide, drop by drop, until a blue color is obtained. 



In the detection of small amounts of blood the quantity of guaiac 

 used should also be decreased. Do any other substances respond in a 

 similar manner to this test? Is a positive guaiac test a sure indication 

 of the presence of blood? (See discussion on page 258.) 



12. Schumm's Modification of the Guaiac Test. To about 5 c.c. of the 

 solution under examination 6 in a test-tube add about 10 drops of freshly prepared 

 alcoholic solution of guaiac. Agitate the tube gently, add about 20 drops of old 

 turpentine, subject the tube to a thorough shaking and permit it to stand for about 

 two to three minutes. A blue color indicates the presence of blood in the solution 

 under examination. In case there is insufficient blood to yield a blue color under 



1 The Scarlet Runner is a familiar variety purchasable in every seed store. It occurs in 

 two varieties, the white and the red. Ricin, a protein constituent of the castor bean, also 

 possesses pronounced agglutinating properties. Because of its poisonous nature it is, how- 

 ever, not suitable for use in class experiments. 



2 Mendel: Archivio di fisiologia, 7, 168, 1909; Schneider: Journal Biol. Chem., n, 47, 

 1912. 



3 Rabbit's blood is especially desirable (Mendel: loc, cit.} and may be obtained for the 

 purpose by bleeding from a small cut on the animal's ear and defibrinating. 



4 Buckmaster advises the use of an alcoholic solution of guaiaconic acid instead of an 

 alcoholic solution of guaiac resin. 



6 Alkaline solutions should be made slightly acid with acetic acid, as the blue end- 

 reaction is very sensitive to alkali. 



