125 



7. Titrate and adjust reaction to neutral. 



8. Heat in Arnold sterilizer for thirty minutes; shake or stir well 

 and heat again for fifteen minutes. 



9. Determine weight and restore loss by evaporation. 



10. Determine volume, titrate, and adjust reaction to desired point 

 (usually one per cent acid). 



11. Heat again for five minutes if adjustment of reaction has been 

 necessary. 1 



12. Filter through absorbent cotton, passing the filtrate through 

 the same filter until clear. 



13. Titrate and record the final reaction. 



Place in cotton-plugged sterile flasks or plugged sterile test tubes, 

 and sterilize for thirty minutes in the Arnold sterilizer on three suc- 

 cessive days, leaving at room temperature in the intervals. 



Sugar-Free Broth. 1. Make 1 liter of meat infusion broth, following 

 steps 1, 2, 3, 4, 5, 6, 7, and 8 2 ; then filter through thin cotton filter to 

 remove gross particles total clearing is not necessary. 



2. Put the broth in a flask and cool. Then add 10 c.c. of a twenty- 

 four-hour broth culture of B. coli communis. 



3. Place the flask, stoppered with cotton, in the incubator at 37 C. 

 for eighteen hours. (The bacteria will ferment and thus destroy any 

 sugar [monosaccharid] which may be present in the broth, and thus 

 render the broth sugar-free and acid.) 



4. Heat thoroughly to kill the bacteria. 



5. Determine weight and bring to 1,015 gms. Then determine 

 volume and titrate, and adjust to neutral. Heat thoroughly again. 



6. Filter through filter paper until clear. 



7. The pure sugars, dextrose, lactose, saccharose, etc., are then added 

 to separate portions (250 c.c.) of the broth in the proportion of one 

 per cent. 



8. When the sugars are dissolved, tube the broth immediately in 

 fermentation tubes, and sterilize by discontinuous sterilization, never 

 heating over twenty minutes at a time, as heat tends to destroy or 

 change the sugars. 



Glycerin Broth. To ordinary, slightly acid or neutral meat infusion 

 broth, add six per cent of C. P. glycerin. Sterilize by fractional method. 



1 Media become more acid on boiling, probably because of a driving out of CO 2 , 

 and a second titration therefore becomes necessary. 



2 These steps refer to the regular directions for making infusion broth. One 

 liter of previously made infusion broth may be used instead. 



