METHOD OF ISOLATION 47 



The broth is made thus : Weigh out 5 grams of Lemccfs 

 extract of meat and 10 grams of fresh Witte's peptone. Dissolve 

 these ingredients in a large flask containing 1000 c.c. of distilled 

 water at ordinary room temperature. 



2. Addition of Gelatine. 



Add to the flask of broth just prepared 100 grams of best 

 sheet gelatine, and allow it to soak for half an hour. Place the 

 flask in hot water in a saucepan and heat slowly until the gelatine 

 and other materials are dissolved, shaking the flask from time to 

 time to mix the contents. 



3. Neutralization process. 



For neutralization the following solutions are required : 



(1) Normal hydrochloric acid. 



(2) i-2oth normal acid. 



(3) Normal caustic soda. 



(4) i -2oth normal caustic soda. 



(5) i gram of phenolphthalein in 100 c.c. of 50 per cent. 



alcohol. 



Take from the flask 5 c.c. of the nutrient gelatine as prepared 

 above (i and 2), add it to 45 c.c. of distilled water in a porcelain 

 dish. Boil three minutes to expel any CO 2 and add to it i c.c. 

 of the phenolphthalein solution. Run into the hot solution from 

 a burette just sufficient of the i-2oth normal caustic soda solution 

 to produce a clear bright pink colour, noting how many cubic 

 centimetres of the soda solution are needed. Repeat again 

 and find the average number of cubic centimetres of soda 

 needed to neutralize 5 c.c. of the nutrient medium. Then 

 calculate how many cubic centimetres will be needed to 

 neutralize the whole of the medium left in the flask. Divide 

 by twenty, this will give the number of cubic centimetres of the 

 normal caustic soda solution which will be needed to neutralize 

 the whole of the medium. Add this amount of normal soda 

 to the medium in the flask and test if it is just neutral, as it 

 ought to be, by taking out a few cubic centimetres and 

 adding to it a drop or two of the phenolphthalein. If it is 



