842 EXPERIMENTAL INFECTION AND IMMUNITY 



fluid with a sterile pipet and plant several loopf uls of bacteria on slants of 

 agar and in neutral bouillon. Why is it advisable to wash the sediment? 

 (e) What advantages has the macroscopic over the microscopic 

 method? 



EXPERIMENT 55. MACROSCOPIC AGGLUTINATION REACTION (KOLLE) 



1. Prepare dilutions in amounts of 1 c.c. of a typhoid immune serum in proper 

 test-tubes, ranging from 1 : 20 up to 1 : 1280. 



2. Add one loopful of a twenty-four-hour culture of Bacillus typhosus to each 

 tube, being careful to emulsify thoroughly on the side of the test-tube according to the 

 technic given. This does not materially alter the degree of dilution. Prepare the 

 culture control as usual. 



3. Incubate and examine tubes as in the previous experiment. 



What are the advantages and disadvantages of this method? 



EXPERIMENT 56. MACROSCOPIC AGGLUTINATION REACTION (KILLED 

 CULTURE) 



1. Inoculate a flask containing 200 c.c. of neutral broth with Bacillus typhosus 

 and incubate for forty-eight hours. At the end of this time a good rich growth is 

 usually secured. Shake gently to stir and break up any clumps of bacilli and heat 

 at 60 C. for one hour on a water-bath, gently shaking the flask once or twice during 

 this time. Add 5 c.c. formalin; shake, and place in the refrigerator for three days; 

 stopper the flask with a rubber stopper, and before using shake well in order thor- 

 oughly to mix the dead bacilli which drop to the bottom of the flask. 



2. Prepare a series of dilutions of typhoid immune serum and conduct this 

 experiment according to the macroscopic technic, using the dead instead of a living 

 bacillary emulsion. 



(a) What are the advantages of using a killed culture? 



(b) Is this method as delicate as when living cultures are used? 



(c) Is spontaneous agglutination likely to occur? 



(d) What constitutes a satisfactory killed culture for this reaction? 



EXERCISE 22. AGGLUTININS (Continued) 

 EXPERIMENT 57. GROUP AGGLUTINATION 



1. Prepare five series of test-tubes containing 1 c.c. of dilutions ranging from 1:20 

 to 1 : 640 of a typhoid immune serum. 



2. To the first series add 1 c.c. of a thirty-six-hour bouillon culture of Bacillus 

 typhosus; to the second series, Bacillus paratyphosus "B"; to the third, Bacillus 

 paratyphosus "A"; to the fourth, Bacillus enteritidis (Gartner); to the fifth, Bacillus 

 coll. The dilutions are thus doubled. Prepare culture controls of all five cultures, 

 and be careful that tubes are properly labeled. 



3. Incubate for two hours and record reactions, at the end of further six hours at 

 room temperature. 



4. This experiment may be repeated with the serum of a typhoid convalescent 

 patient. 



