PRACTICAL EXERCISES 65 



them ' Magnesium Sulphate A, B, C, D.' Dilute B with four times, C 

 with eight times, and D with twenty times as much distilled water as 

 was taken of the plasma. Observe in which, if any, coagulation occurs, 

 and the time of its occurrence, and report the result. 



(n) With peptone plasma from H and from the peptone blood 

 obtained later repeat the experiments done in (7). In addition dilute 

 i c.c. of the plasma with three volumes of water and i c.c. of it with 

 ten volumes of water, and put in the bath at 40 C. Observe whether 

 clotting occurs. 



Instead of dog's blood, the blood of an ox or pig may be obtained at 

 the slaughter-house. 



4. Preparation of Fibrin-Ferment. Precipitate blood-serum with 

 ten times its volume of alcohol. Let it stand for several weeks, then 

 extract the precipitate with water. The water dissolves out the fibrin- 

 ferment, but not the coagulated serum proteins. 



5. Preparation of Tissue Extracts containing Thrombokinase. In a 

 dog or rabbit killed by bleeding insert a cannula into the lower end of 

 the thoracic aorta. !Fill the cannula with 0*9 per cent, salt solution, 

 and connect it with a bottle also containing salt solution. Wash 

 put the vessels of the lower portion of the body, making an opening 

 in the inferior vena cava above the diaphragm to allow the liquid 

 to escape. For the sake of cleanliness, a cannula armed with a 

 piece of rubber tubing should be inserted for this purpose into the 

 inferior vena cava. Continue the injection till the liquid issues colour- 

 less. Then remove portions of liver and muscle. Mince each separately. 

 Rub up with sand in a mortar. Add 0-9 per cent, sodium chloride 

 solution and rub up again. Put into bottles and keep in the ice-chest. 

 For use take off some of the liquid from the top with a pipette, or strain 

 through cheese-cloth. 



6. Serum. Test the reaction, and determine, both by the hydrom- 

 eter and the pycnometer, or specific gravity bottle, the specific 

 gravity of the serum provided, or of the serum obtained in experi- 

 ment 3. 



Serum Proteins. (i) Saturate serum with magnesium sulphate 

 crystals at 30 C. The serum-globulin is precipitated. Filter off. 

 Wash the precipitate on the filter with a saturated solution of mag- 

 nesium sulphate. Dissolve the precipitate by the addition of a little 

 distilled water, and perform the following tests for globulins : (a) Satu- 

 rate with magnesium sulphate. A precipitate is obtained, (b) Drop 

 into a large quantity of water, and a flocculent precipitate falls down. 

 (c) Heat. Coagulation occurs. Determine the temperature of coagu- 

 lation (p. 9). 



(2) To a portion of the filtrate from (i) add sodium sulphate to 

 saturation. The serum-albumin is precipitated. (Neither magnesium 

 sulphate nor sodium sulphate precipitates serum-albumin alone, but 

 the double salt sodio-magnesium sulphate precipitates it, and this is 

 formed when sodium sulphate is added to magnesium sulphate.) 



(3) Dilute another portion of the filtrate from (i) with its own bulk 

 of water. Very slightly acidulate with dilute acetic acid, and de- 

 termine the temperature of heat coagulation. 



(4) Precipitate the serum-globulin from another portion of serum by 

 adding to it an equal volume of saturated solution of ammonium 

 sulphate. Filter. Precipitate the serum-albumin from the nitrate by 

 saturating with ammonium sulphate crystals. 



(5) Dilute serum with ten to twenty times its volume of distilled 

 water, and pass through it a stream of carbon dioxide. The serum- 

 globulin is partially precipitated. 



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