AGGLUTININS 917 



2. Using a good twenty-four-hour-old culture of Bacillus typhosus, prepare an 

 agglutination test after the technic given hi the text-book. Be particularly careful 

 not to transfer paper fiber to the slide apply the salt solution and dissolve the blood 

 by gently rubbing with a small platinum loop (2 mm.). Mix the blood in the loop- 

 ful of culture with the slide held or placed over a white surface so that the proper 

 delicate orange tint is secured. 



3. Prepare culture control as usual; also similar tests with a known positive and 

 negative serum. 



4. Examine at the end of an hour. 



(a)' What special precautions are to be observed in this method? 



(b) What is the particular practical value of this reaction? 



(c) Are accurate dilutions possible and if so, by what technic? 



(d) Are agglutinins resistant to deleterious influences? 



(e) Compare the value of this method with the one used in the pre- 

 ceding experiment. 



EXERCISE 2 J. AGGLUTININS (Continued) 

 EXPERIMENT 54. MACROSCOPIC AGGLUTINATION REACTION 



1. Secure serum from a rabbit which has been immunized with Bacillus typhosus. 

 Serum of a typhoid convalescent may be used instead. 



2. Prepare a series of serum dilutions in small narrow test tubes in amounts of 

 1 c.c., ranging from 1: 10 up to 1:640. 



3. Add to each 1 c c. of the bacillary emulsion of a good twenty-four- to forty- 

 eight-hour bouillon culture of Bacillus typhosus or an emulsion prepared by washing 

 twenty-four-hour growths from agar slant cultures with normal salt solution, accord- 

 ing to the technic given in the text. This doubles the dilutions, which now range 

 from 1 : 20 up to 1 : 1280. Prepare the culture control. 



4. Shake gently and incubate for two hours at 37 C. and then record results after 

 tubes have been standing at room temperature for six hours. Reexamine tubes with 

 the agglutinoscope and note the higher delicacy of such readings. 



5. If a typhoid immune serum of unknown titer is used and agglutination is 

 complete in the highest dilution, the test must be repeated with still higher dilutions 

 in order to determine the agglutinin titer of the serum. 



(a) Has agglutination occurred in the control tube? Why is this 

 control so important in this and all agglutination reactions? 



(b) Is agglutination as complete in the lower as in the higher reac- 

 tion? If not, how do you explain this result? Of what practical im- 

 port is this phenomenon? 



(c) Describe the appearance of macroscopic agglutination. 



(d) Are the agglutinated bacilli dead? To determine this, pipet off 

 the supernatant fluids of several tubes into a germicidal solution; then 

 add an excess of sterile normal salt solution to the sediment of aggluti- 

 nated bacilli, stir up the sediment, and transfer with a sterile pipet to a 

 sterile centrifuge tube; centrifuge thoroughly; remove the supernatant 



