GENERAL BACTERIOLOGY 



A. 



1.) Titrate as follows: Pipette off 5 

 cc. of the fluid into a 10 cm. evaporating 

 dish, add 45 cc. of distilled water, boil 

 for three minutes, add 1 cc. of phen- 

 olphthalein (0.5% substance in 50% al- 

 cohol), and then run in carefully, drop 

 by drop, from a burette a twentieth 

 normal 1 solution of sodium hydroxide 

 (^NaOH) until the solution turns a 

 faint pink color. Treat two other sam- 

 ples in the same way. If the amount of 

 NaOH required is approximately the 

 same in each case the average can be 

 taken as the amount necessary to neu- 

 tralize 5 cc. Calculate the amount nec- 

 essary to neutralize the whole (100015 

 cc.). Since this amount would dilute 

 the medium too much, a stronger solu- 

 tion (normal) is used, hence, 



Use a normal 1 solution of so- 

 dium hydroxide (NaOH). Add 

 to the hot solution a few cc. at a 

 time at first, later a few drops, 

 stirring thoroughly with a glass 

 rod. After each addition, test 

 by placing a drop of the solution 

 by means of the glass rod on a 

 strip of phenolphthalein paper. 

 (Prepared by dipping filter pa- 

 per in a solution.) The addition 

 should continue until the test pa- 

 per is turned a faint pink color. 



2.) Neutralize by adding -gVtli of the volume calculated above 

 of a normal solution of sodium hydroxide. Test the accuracy of 

 the work at this point by the addition of a few drops of phenol- 

 phthalein to a cc. or so of the medium. If a faint pinkish tint is 

 not obtained, titration and neutralization must be repeated. 



g. Boil for 5 minutes and restore weight. 



h. Test reaction and adjust if necessary. 



i. Add 0.5 to 1.5% of a normal hydrochloric acid. The amount 

 of acid to be added varies with the purpose for which the medium 

 is to be used, e. g., in water analysis + 1.0 (acid) is preferable, 

 with the pathogenic bacteria a smaller amount of acid (J-0.5) 

 more nearly meets requirements. 



j. Heat until precipitate appears flaky and then filter through 

 moistened filter paper. (For method of folding see Abbott p. 96). 



The filtrate (bouillon) should be of a light straw color, per- 

 fectly clear, and should not give a precipitate on boiling. 



REFERENCES. A. 94; H. 45; M. & B. 35; McF. 180; P. 212; 

 P. B. C. 18-24. 



SPECIAL DIRECTIONS. Prepare 1 liter of bouillon according to 

 method C. Secure and put to soak meat for 7. See Rule IV. 



1 Normal solutions are prepared so that one liter at 16 C. shall contain 

 the hydrogen equivalent of the active reagent weighed in grams (Sutton). 

 For present purposes a 4% solution of sodium hydrate is sufficiently accurate. 



