838 EXPERIMENTAL INFECTION AND IMMUNITY 



EXERCISE 18. FERMENTS AND ANTIFERMENTS 

 EXPERIMENT 49. TRYPTIC FERMENT OF LEUKOCYTES 



1. Collect 0.5 c.c. of blood in each of two small test-tubes by puncture of a finger; 

 set aside until coagulation has occurred. Add several changes of warm distilled water 

 until the red corpuscles are hemolyzed and colorless clots of leukocytes, fibrin plate- 

 lets, and detritus are secured. 



2. Place one clot in the bottom of a tube of sterile and slanted Loeffler blood- 

 serum medium which is fairly dry and firm. 



3. Place the second clot in a second tube of this medium and add 0.2 to 0.4 c.c. of 

 fresh serum. 



4. Plug these tubes firmly, paraffin the stoppers to prevent evaporation, and 

 incubate along with a non-inoculated control at 50 C. for two days. 



(a) In which tube do you note evidences of digestion? 



(b) Describe the appearance of the digested medium. 



(c) Does the tube containing serum show digestion? How do you 

 explain the result? 



(d) What role may this ferment play in infection? 



(e) Why do not the leukocytes digest themselves? 



(f) What is the nature of the ferment? 



(g) May this ferment play a r61e in the disposal of old and dead 

 leukocytes? 



EXPERIMENT 50. TESTING THE ANTITRYPTIC POWER OF BLOOD- 

 SERUM (AFTER THE MARCUS MODIFICATION OF THE METHOD OF 

 MtJLLER AND JOCHMANN) 



1. Prepare a solution of trypsin by thoroughly shaking 0.1 gm. of Kahlbaum's 

 trypsin with 5 c.c. glycerin and 5 c.c. distilled water. Incubate at 55 C. for half an 

 hour, shake thoroughly, filter, and preserve in the refrigerator. 



2. Secure six Petri dishes of Loeffler blood-serum culture-media which have been 

 sufficiently dried to drive off the water of condensation and with a firm elastic surface. 



3. Collect 1 c.c. of blood from a patient three hours after last meal; separate the 

 serum. 



4. In a watch-glass or hanging-drop slide mix one drop of serum with an equal 

 sized drop of trypsin solution. Mix well and transfer five loopfuls to an area on a 

 Petri dish of medium. With a blue-wax pencil draw a circle on the cover of the plate 

 to include the site of planting and mark No. 1. 



5. Prepare serial dilutions with one drop of serum with two, three, four, five, six, 

 seven, eight, nine, and ten drops of trypsin solution. Mix well and plant five loop- 



ils of each dilution on the medium in five dishes. Two plantings may be made in each 

 plate and with care they will not become confluent. Mark each plate. 



6. Inoculate the sixth plate with five drops of trypsin solution without serum 

 (control). 



7. Incubate all tubes at 37 C. for six to twelve hours until the control shows 

 well-marked digestion. 



8. Wherever the trypsin has not been neutralized by the serum, shallow liquefied 



