TOXINS 893 



5. Observe all animals for forty-eight hours, taking the rectal temperature night 

 and morning. Make leukocyte counts every four hours during the day. Make 

 physical examination of the chest. 



6. Autopsy the animals under aseptic precautions and with complete anesthesia. 



7. Culture the heart's blood of each in serum bouillon. 



8. Culture the pulmonary lesions in serum bouillon. 



9. Prepare smears of the heart's blood and lesions and stain with methylene-blue 

 and Gram's stain. 



10. Remove consolidated portions of lung and place in 5 per cent, formalin. 

 After twenty-four hours, cut sections which are passed through by the paraffin 

 method and stained with hematoxylin and eosin, methylene-blue, and Gram's stain. 



(a) Did any of the animals show evidences of infection? 



(b) Are there evidences of pneumonia? How do these lesions com- 

 pare with those of human pneumonia? 



(c) How do you explain their production? 



(d) Does the animal receiving the smaller dose of pneumococci intra- 

 bronchially show evidences of pneumonia? If not, why not? Does 

 this show a numerical relationship of bacteria to infection? 



(e) Were the temperature changes similar to those observed in 

 human lobar pneumonia? 



(f) Did leukocytosis occur and if so, why? 



(g) Are there any evidences of pleuritis and if so, how do you explain 

 its production? 



(h) Did the dog receiving the pneumococci intravenously show evi- 

 dences of pneumonia? Does this bear any relation to the question of 

 the route of introduction of bacteria to infection? 



(i) Are the pneumococci seen in the smears of the lesions and blood 

 encapsulated? If so, what is the significance of these capsules? Com- 

 pare these cocci with those shown in the smear of the culture before in- 

 jection? Are the capsules lost in the artificial culture-media? 



(j) Discuss the question of the possible modes of infection in human 

 lobar pneumonia, especially inspiratory pneumonia. 



EXERCISE 3. TOXINS 



EXPERIMENT 6. DIPHTHERIA TOXIN 



1. Inoculate tubes of neutral or slightly alkaline bouillon with a virulent culture 

 of diphtheria bacilli (Park-Williams Bacillus No. 8 being especially desirable). 



2. Grow at 35 C. for five days and filter the culture through a Berkefeld filter. 



3. Inject a 250- to 300-gram guinea-pig in the median abdominal line with 0.5 

 c.c. of the filtrate (toxin). 



4. Heat 1 c.c. of toxin at 60 C. for an hour and inject 0.5 c.c. into a second guinea- 

 pig subcutaneously. 



