2 JO PHYSIOLOGICAL CHEMISTRY 



to remove hemoglobin. Squeeze out the water, mince the fibrin and cover with 

 an 8 per cent sodium chloride solution and allow to stand in the cold for 48 hours. 

 Filter. Precipitate the thrombin (and other proteins) from the filtrate by adding 

 an equal volume of acetone. Filter the mixture rapidly through a number of 

 small (25-50 c.c.) filters. Spread out filter papers and precipitate and dry rapidly 

 in a current of cold air. Cut the dried papers into small pieces and treat with a 

 volume of water equivalent to 66 per cent of the 8 per cent NaCl previously used. 

 Allow to stand one-half hour and filter. Shake the filtrate with chloroform 

 (10-15 c.c. per 100 c.c. filtrate) until on settling no opalescence is developed by 

 heating a portion of the supernatant fluid. Decant the liquid and evaporate 

 on watch glasses (2 c.c. to a watch glass) in a current of air. Thrombin so pre- 

 pared may be kept indefinitely in a desiccator. 



19. Variation in Size of Erythrocytes. Prepare two small funnels with filter 

 papers such as are used in quantitative analysis. Moisten each paper with physio- 

 logical (isotonic) salt solution. Into one funnel introduce a small amount of 

 denbrinated ox blood and into the other funnel allow blood to drop directly from a 

 decapitated frog. Note that the nitrate from the ox blood is colored, whereas that 

 from the frog blood is colorless. What deduction do you make regarding the 

 relative size of the erythrocytes in ox and frog blood? Does either nitrate clot? 

 Why? 



II. Blood Serum 1 



1. Coagulation Temperature. Place 5 c.c. of undiluted serum in a test-tube 

 and determine its temperature of coagulation according to the method described 

 on page 104. Note the temperature at which a cloudiness occurs as well as the 

 temperature at which coagulation is complete. 



2. Precipitation by Alcohol. To 5 c.c. of serum in a test-tube add twice the 

 amount of 95 per cent alcohol and thoroughly mix by shaking. What is this pre- 

 cipitate? Make a confirmatory test. Test the alcoholic filtrate for protein. 

 Explain the result. 



3. Proteins of Blood Serum. Place about 10 c.c. of serum in a small evapo- 

 rating dish, dilute with 5 c.c. of water and heat to boiling. At the boiling-point 

 acidify slightly with dilute acetic acid. Of what does this coagulum consist? 

 Filter off the coagulum (reserve the filtrate) and test it as follows : 



(a) Millon's Reaction. Make the test according to directions given on page 



97- 



(b) Hopkins-Cole Reaction. Make the test according to directions given on 



page 98. 



4. Sugar in Serum. To 5 c.c. of the neutralized filtrate from Experiment 3 

 add 5 drops of Fehling's solution and boil one minute. What do you conclude? 



5. Detection of Sodium Chloride. (a) Test a little of the filtrate from Ex- 

 periment 3 for chlorides, by the use of nitric acid and silver nitrate, (b) Evapo- 

 rate 5 c.c. of the filtrate from Experiment 3 in a watch glass on a water-bath. 

 Examine the crystals and compare them with those reproduced in Fig. 86, p. 269. 



6. Separation of Serum Globulin and Serum Albumin. Place 10 c.c. of blood 

 serum in a small beaker and saturate with magnesium sulphate. What is 

 this precipitate? Filter it off and acidify the filtrate slightly with acetic acid. 

 What is this second precipitate? Filter this precipitate off and test the filtrate by 

 the biuret test. What do you conclude? 



1 For directions as to preparation of serum, see "Reagents and Solutions." (Page 631.) 



m * 



