74 BACTERIOLOGICAL METHODS 



If streaming or live steam is to be used in place of the steam 

 under pressure in the autoclave, intermittent sterilization is to 

 be practised. Place the media in the steam sterilizer for 30 

 min. on each of 3 successive days. Wait until the tem- 

 perature in the sterilizer has risen to approximately 100° C. 

 before placing the media therein. Agar media should first be 

 liquefied. At the end of each period, remove the media and cool 

 as rapidly as possible for reasons already given. 



When media are prepared under the proper laboratory condi- 

 tions and sterilized as above suggested, they are as a rule free from 

 all living germs. However, if practicable, the media should be 

 watched for a period of 2 days, stored in a room at ordinary 

 temperature, in order to note possible bacterial developments. 



3. Adjustment of Reaction of the Media. — As a rule bacteria 

 develop most actively in media which are slightly alkaline to 

 litmus and since certain media are quite acid in reaction (gelatin 

 in particular) it becomes necessary to reduce them to a standard 

 reaction. The standard indicator to be used is phenolphthalein. 

 When phenolphthalein is not obtainable, Htmus paper (or a i 

 per cent, aqueous solution of Kahlbaum's azolitmin) may be 

 used. The reaction adjustments are to be made as follows: 



Place 5 cc. of the medium to be tested in 45 cc. of distilled 

 water (making a dilution of i-io). Boil briskly for i min., 

 with stirring or rotary shaking. Add i cc. of the phenolphthalein 

 solution (made by dissolving 5 grams of the salt in i liter of 

 50 per cent, alcohol). Titrate while hot with N/20 caustic 

 soda solution (in distilled water). A distinct pink coloration 

 marks the proper reaction. To be more precise, the pink should 

 correspond to a mixture or combination of 25 per cent, red and 



75 per cent, white of the color top recommended by the Com- 

 mittee on Standard Methods of the American Health Associa- 

 tion. The reactions of the media are stated in terms of the 

 percentages of normal acid or alkaline solutions required to 

 neutralize them. Alkalinity is indicated by the minus ( — ) 



