QO SOIL BACTERIOLOGY 



8. Sterilize broth in large flask and allow to stand until 

 next laboratory period. 



9. Refilter through fine paper and tube. 



10. Fill tubes about one-third full. 



11. Plug the tubes and sterilize in autoclave at 120 C. 

 for fifteen minutes. 



Note. To titrate, remove 5 c.c. of the medium to a casserole or small 

 flask containing about 45 c.c. of distilled water. Boil one minute with 

 constant stirring, add 3 drops phenolphthalein, and neutralize excess acid 

 with N/20 NaOH. If i c.c. N/20 NaOH is required to neutralize 5 c.c. 

 of the medium, the reaction is correct. In this way calculate the amount 

 of normal alkali or acid necessary to adjust the reaction of the entire bulk 

 of culture-medium to i per cent. (+i). 



All reactions must be expressed with reference to the phenolphthalein 

 neutral point. They are stated in percentages of normal acid or alkaline 

 solutions required to neutralize them (Fuller's scale). Alkaline media 

 should be recorded with the minus sign ( ) before the percentage of normal 

 acid needed for their neutralization; acid media should be written with the 

 plus sign (+) before the percentage of normal alkaline solution necessary 

 for their neutralization. 



The example below will illustrate the method. If the required reaction 

 is (+1) and the buret reading shows that i.S c.c. of N/20 NaOH has been 

 used in neutralizing the 5 c.c. of broth, then the problem may be stated as 

 follows: 



5 c.c. of broth require the addition of 1.8 c.c. N/20 NaOH to neutralize 

 it. 



100 c.c. of broth require the addition of 36 c.c. N/2O NaOH or 1.8 c.c. 



N/i NaOH to neutralize it. 



looo c.c. of broth require the addition of 18 c.c. N/i NaOH to neutralize 

 it. 



The figures above show that the broth as titrated is 0.8 per cent, too 

 acid, and that 8 c.c. of normal NaOH per liter must be added to obtain the 

 proper reaction. 



Do not neutralize medium first and then readjust by the addition of acid. 

 This tends to precipitate certain substances which are favorable to bac- 

 terial development. 



The broth prepared in this way should be of a golden color and 

 should not develop a precipitate upon subsequent sterilization in the 

 autoclave. Adjust reaction by adding normal hydrochloric acid or 

 sodium hydroxid. 



