BACTERIOLYSIS 939 



EXERCISE 50. BACTERIOLYSIS 



EXPERIMENT 103. MICROSCOPIC METHOD OF MEASURING THE BAC- 

 TERIOLYTIC POWER OF THE BLOOD 



This method may be employed for a rapid estimation of the bac- 

 teriolysin produced in the blood in response to an inoculation of typhoid 

 vaccine. 



1. Secure a small quantity of the patient's serum by collecting blood aseptically 

 in a Wright capsule. About 1 c.c. of serum will be sufficient. Secure a control 

 serum, preferably a "pooled serum," in the same manner. Prepare dilutions of the 

 patient's serum in the following manner: Place a series of six small test-tubes (sterile) 

 in a rack; add 1 c.c. sterile broth to each tube; into the first tube place 1 c.c. of the 

 fresh serum from the patient (1:2 dilution) ; mix well and transfer 1 c.c. to the second 

 tube; mix and transfer 1 c.c. to the third, and so on to the last tube, when 1 c.c. is 

 discarded. 



2. Secure a twenty-four-hour culture of typhoid bacilli in neutral bouillon. 



3. Take a simple capillary pipet with a long stem, plugged with cotton and steril- 

 ized, and make a mark about 3 cm. from the end. Draw up the highest dilution of 

 serum to the pencil mark, then a bubble of air, and finally an equal volume of culture. 

 Thoroughly mix by carefully aspirating and driving out the contents on a hollow 

 ground slide or in a small tubule. Aspirate the mixture into the middle portion of 

 the stem and seal the pipet in a flame. Label with the final dilution (1 : 128). 



4. Proceed in the same manner with the remaining five dilutions of the patient's 

 serum, which then, mixed with an equal quantity of culture, equals 1 : 64, 1 : 32, 1 : 16, 

 1 : 8, and 1 : 4. Place these pipets in a large test-tube and label with the patient's 

 name and time when placed in the incubator. 



5. Prepare a similar set of pipets using the control serum. 



6. Prepare a culture control by mixing equal volumes of culture and sterile broth. 



7. Place all pipets in the incubator at 37 C. for two hours. 



8. Prepare hanging-drop preparations of each pipet by breaking off the tip and 

 placing a drop of the contents (after mixing) on a cover slide and suspending in the 

 usual manner. First examine the culture control and then each of the various dilu- 

 tions, noting in each case the dilution in which there is the first bacteriolytic effect, 

 and the dilution in which there is a complete effect; arrive at the bacteriolytic index 

 by comparing the patient's and the control blood just as the opsonic index is stated. 



(a) What are the first evidences of bacteriolysis? 



(b) Are endotoxins neutralized when bacteriolysis occurs? 



EXERCISE 5f. BACTERIOLYSIS 



EXPERIMENT 104. METHOD OF MEASURING THE BACTERICIDAL AC- 

 TIVITY OF THE BLOOD IN VITRO (METHOD OF STERN AND KORTE) 



With a culture of typhoid bacilli, rabbit typhoid immune serum, and rabbit 

 complement serum, a test may be carried out after the method given in the text. 



(a) What main precautions are to be followed in this method? 



(b) Discuss complement deviation. 



(c) Discuss the practical value of this method. 



