BACTERIOLOGICAL TECHNIC 19 



cent more alkaline than the neutral point of phenolphthalein, 

 so that a medium which is neutral to phenolphthalein is about 

 2 per cent alkaline to litmus. It has been found that about i 

 per cent acid to phenolphthalein is the most favorable reaction 

 for the growth of pathogenic bacteria. A medium of this reac- 

 tion is still alkaline to Htmus. 



7. After neutralization fill into an Erlenmeyer flask 

 and autoclave for 10 minutes at 120° C. 



8. Keep the sterilized broth for 24 hours and then 

 filter until clear and distribute into culture tubes, 

 which have to be autoclaved again. 



Note. — ^The reason for exposing broth to a heat of 120° C. 

 twice is this: The solution contains substances which are pre- 

 cipitated by heat and appear as a sediment after cooling. As it 

 is important to have a perfectly clear broth in tubes, these sub- 

 stances are precipitated by the first heating, and, if tubed later, 

 the second sterilization will not affect the appearance of the 

 medium. 



For ordinary purposes it is sufficient to neutralize media by 

 means of phenolphthalein paper. This is prepared by soaking 

 filter paper in a 1 per cent solution of phenolphthalein in 5° per 

 cent alcohol and then allowing the paper to become dry. A 2 per 

 cent or 4 per cent solution of sodium hydrate is added to the' 

 medium to be neutralized until a faint, but decided, pink appears 

 on phenolphthalein paper. 



A more precise method is as follows: Measure by means of a 

 pipette s c.c. of the medium into a white porcelain evaporating 

 dish, add 45 c.c. of distilled water and i c.c. of a i per cent solu- 

 tion of phenolphathalein in 5° per cent alcohol. Heat the mix- 

 ture to boiling and slowly add from a graduated burette i-2oth 

 normal NaOH imtil a faint but decided and stable pink appears 

 in the liquid. The amount of NaOH is read from the burette 

 and the amount for neutralization of the whole volume calculated. 

 It is desirable to make another titration after the NaOH has 

 been added. The amount to be added to the medium has to be 

 varied according to the reaction desired. If it is to be neutral, 



