IMPORTANT PATHOGENIC BACTERIA 125 



Stain film for 2 seconds with solution a and wash. 

 Apply solution b for 3 to 5 seconds and wash well. 

 Apply solution c for 3 to 5 seconds, wash, dry, mount. 



3. Special study. — Test for acid formation in a 

 culture, in neutral glucose broth, one week old, by addi- 

 tion of a few drops of litmus solution, or by titration 

 with N-^ NaOH solution. 



4. Special study. — Cultivation of B. diphtheriae 

 on eggs (method of Wyatt Johnston). 



Note. — This method is recommended as an emergency cul- 

 ture test, the egg taking the place of Loffler's blood serum. 



a) Sterilize over the flame an empty meat extract 

 pot, or any other vessel of suitable size. 



b) Wash a hard-boiled egg in mercuric chlorid 

 solution, followed by alcohol, and break the shell at the 

 blunt end with sterile forceps, without rupturing the 

 membrane lining the shell. 



c) Flame the exposed part, and free the coagulated 

 albumen from the membrane. 



d) Inoculate by rubbing some culture or throat 

 swab on the exposed egg-albumen. 



e) Invert and set in the sterilized pot. 

 /) Incubate at 37° C. 



g) Observe the appearance and make a stained 

 preparation after 24 hours. 



5. Special study — 

 Experiment i — 



a) CUp the hair over a small area on the surface of 

 the abdomen of a guinea-pig or rabbit. 



b) Cut a small opening in the skin, and separate the 

 skin from the muscles below by pushing in sterile 



