General Bacteriology. 



B. 



Use a normal solution of so- 

 dium hydroxide (yNaOH). 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 solu- 

 tion by means of the glass rod 

 on a strip of red litmus paper, 

 and then moisten the paper with 

 distilled water. The addition 

 should continue until the red 

 litmus paper is turned blue, but 

 no change occurs on blue litmus 

 paper. 



A. 



1 . ) Titrate as follows : Pipette off 5 cc: of the fluid 

 into a 4-inch evaporating dish, add 45 cc. of distilled 

 water, boil for three minutes, add 1 cc. of phenol- 

 phthalein (0.5% substance in 50% alcohol), and then 

 carefully run in, drop by drop, from a burette a twen- 

 tieth normal * solution of sodium hydroxide (^VNa 

 OH) until the solution turns a faint pink color. Treat 

 two other samples in the same way. If the amount 

 of Na OH required is approximately the same in each 

 case the average can be taken as the amount necessary 

 to neutralize 5 cc. Calculate the amount necessary 

 to neutralize the whole (1000-15 cc.). Since this 

 amount would dilute the medium too much, a stronger 

 solution (normal) is used, hence, 



2.) Neutralize by adding ^Vth of the volume cal- 

 culated above of a normal solution of sodium hydrox- 

 ide. Test the accuracy of the work at this point by 

 the addition of a few drops of phenolphthalein to a 

 cc. or so of the medium. If a faint pinkish tint is not 

 obtained, titration and neutralization must be re- 

 peated. 



If by mistake more alkali is added than is required, the reaction can be corrected by 

 the use of a normal solution of hydrochloric acid. 



h. Boil for 5 minutes and restore weight. 



i. Test reaction and adjust if necessary. 



j. Add 0.5 to 1.5% of a normal hydrochloric acid if neutralized by method A, oth- 

 erwise omit. The amount of acid to be added varies with the purpose for which the 

 medium is to be used, e. g., in water analyses +1.5 (acid) is preferable, with the path- 

 ogenic bacteria a smaller amount of acid (+ 0.5) more nearly meets requirements. 



k. Filter through moistened filter paper (Abbott p. 96), or absorbent cotton, 

 (VII. m). If the filtrate is not perfectly clear, cool to 60 C., add the white of an 

 egg, thoroughly mix and boil for 5 minutes without stirring. 



The filtrate (bouillon) should be of a light straw color, perfectly clear, and should 

 not give a precipitate on boiling. 



REFERENCES. A. 90; M. & R. 43; McF. 124; N. 234; 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 VII. 



EXERCISE V. FILLING TEST-TUBES AND FLASKS WITH CULTURE MEDIA. 



GENERAL DIRECTIONS. In filling tubes be careful not to allow the media to touch 

 the neck of the vessels as this will cause the cotton to stick to the glass when the plugs 

 are removed. Place the culture fluid to be tubed in a funnel arranged with a delivery 



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. 



