APPENDIX 359 



Method. 1. Heavily inoculate a tube of sterile broth 

 with B. coli and incubate at 37 C. 



2. Soak 1 Ib. finely chopped lean beef in 1000 c.c. dis- 

 tilled water over night (twenty-four hours). 



3. Strain out the meat juice and make up to 1000 c.c. 

 with distilled water. 



4. Pour the entire contents of the twenty-four-hour 

 broth culture of B. coli into the meat juice and 



5. Incubate at 37 C. for twelve to sixteen hours, not 

 longer. B. coli uses the fermentable substances, inosite 

 (muscle sugar), dextrose, etc., as food, leaving the meat 

 juice free from fermentable substances. // this action is 

 allowed to proceed too long, poisonous decomposition products 

 of the^ proteins are formed which will inhibit the growth of 

 other microorganisms. 



6. Mix the peptone (1%) into a thin paste with as little 

 water ' as possible and add to the twelve or sixteen-hour 

 culture of B. coli in the meat juice. 



7. Heat in the autoclav for twenty minutes or in the 

 steam for one hour. 



8. Titrate and make neutral to phenolphthalein. 



9. Boil over a free flame for three to five minutes. 



10. Add 1% dextrose and 10 c.c. of 0.5% solution of 

 neutral red and stir until sugar is dissolved. 



11. Filter until clear. 



12. Fill ten fermentation tubes for each student. 



13. Sterilize in autoclav or in flowing steam. 



14. Other sugar broths are prepared by adding instead 

 of dextrose, 1% of the sugar desired. 



Practically all sugar-fermenting organisms will ferment 

 monosaccharides such as dextrose; comparatively few will 

 ferment the disaccharides lactose, saccharose, etc. B. 

 coli will ferment all three sugars to a greater or less extent. 

 Bacteria of the typhoid group ferment none of the three and 

 those belonging to the paratyphoid group ferment dex- 

 trose but not lactose, therefore the use of lactose in culture 



