52 APPLIED BACTERIOLOGY 



The mixture is now boiled for five minutes, and then 

 very carefully neutralised with a solution of sodium car- 

 bonate, making the solution very faintly alkaline to litmus- 

 paper. The alkaline solution is added carefully, drop by 

 drop, shaking the flask well between each addition. The 

 solution is again boiled for ten minutes, with constant 

 agitation. The reaction is again tested with litmus-paper, 

 and if still faintly alkaline, the solution is filtered into a 

 flask through a double-pleated filter-paper. The filtered 

 product, which should be absolutely clear and bright,* 

 is then run into flasks (which are plugged with sterile 

 cotton-wool and covered with tinfoil), and sterilised on 

 three successive days in the steamer for fifteen or twenty 

 minutes on each occasion. 



When neutralising nutrient media under certain circum- 

 stances, depending upon the relative proportions of the 

 different phosphates of potassium and sodium present, 

 what is known as the amphoteric reaction is obtained — i.e., 

 red litmus is turned blue, and blue litmus red by the same 

 solution. Owing to this difficulty, and that due to the 

 varying sensitiveness of different samples of litmus-paper, 

 making the true neutralisation point somewhat indefinite, 

 we prefer the use of phenol-phthalein as indicator. The 

 procedure is as follows : 



An aliquot portion of the nutrient medium is taken, say 

 20 c.c. ; this is diluted with warm distilled water, a few 

 drops of a solution of phenol-phthalein added, and normal 

 solution of sodium hydrate added drop by drop from a 

 burette until a pink coloration is obtained. The correct 

 volume in c.c. of normal soda solution to be added to the 



* If in spite of filtration the broth remains turbid, half the white of an 

 egg is added to the cooled broth, well mixed, then raised and maintained 

 at the boiluag-point for ten minutes. The precipitated albumen is then 

 filtered off and the filtrate sterilized as above directed. 



