46 PURE CULTURE OF BACTERIA 



of the dried plugged tubes in a wire cage and heat in the hot-air 

 sterilizer to 150 C. for one hour (Fig. 15). 



(2) Test-tubes which have been used and contain nutrient 

 gelatine or other media must be dipped in a saucepan of hot 

 water to liquefy the gelatine. Pour out the contents and then 

 place the tube in a saucepan containing a weak solution of 

 caustic potash, tilting them so that they may be completely 

 filled with the solution without imprisoning air bubbles. Boil 

 for an hour. Take out the tubes and wash them in water several 

 times, clean with nitric acid and alcohol and sterilize as explained 

 above, plugging with cotton-wool as with unused new tubes. 



(3) Flasks, beakers, Petri dishes, pipettes, and other glass 

 apparatus should be cleaned and sterilized in the hot-air oven in 

 similar manner. All smaller apparatus and pairs of Petri dishes 

 should be wrapped separately in paper before sterilization. 

 They should be stored in a cupboard or drawer away from dust. 



Ex. 17. The next step is the manufacture of a sterilized 

 nutrient medium with addition of gelatine or agar-agar. This 

 requires great care if reliable and consistent results are to be 

 obtained. Bacteria are very sensitive to acids and alkalis ; unless 

 the media are suitable in this respect the organisms will not 

 grow. It is found that most bacteria grow satisfactorily when 

 the medium contains i per cent, of free normal acid, as ex- 

 plained below, though some commonly met with in the soil 

 require a neutral medium or one which is slightly alkaline. The 

 process of the manufacture of the nutrient gelatine may be con- 

 veniently divided into four stages. 



1. Prepare the nutrient medium (bouillon or broth). 



2. Add gelatine. 



3. Neutralize carefully and completely, using a solution of 

 phenolphthalein in alcohol as indicator. 



4. Add sufficient acid to bring the whole to the proper state 

 of acidity. 



Preparation of Standard Nutrient Gelatine, 

 i . The broth or mttrient medium. 



