PREPARATION OF MEDIA 87 



solution, neutral litmus being used as an indicator. When it 

 gives a good neutral violet color with the litmus it is heated 

 at 100 C. for one hour; thereby nearly all of the proteid is 

 coagulated. It is filtered clear, and neutral litmus is added 

 to a color convenient for observation. 



Neutral red. This dye is added to the peptone and 

 bouillon-sugar media to the amount of 1 to 5 per cent of a 

 concentrated solution. Its reduction * by the growth of 

 bacteria is a valuable aid in the differentiation of certain 

 species. 



Preparation of nitrate bouillon. Take peptonized bouil- 

 lon 200 cc. Add potassium nitrate 1 gram (0.5%). Dissolve 

 the nitrate in the bouillon, put in tubes, and sterilize the 

 same as bouillon. 



The nitrate of sodium or ammonium may be substituted 

 for that of potassium. The salt may be added in the propor- 

 tion of from 0.1 to 1% to meet special demands. 



* Test for nitrites. Twenty-five cubic centimeters of distilled 

 water are added to one-half gram (more or less) of pure potato 

 starch and the fluid boiled. One cubic centimeter or more of this 

 starch-water and 1 cc. of freshly prepared potassium-iodide water 

 (1 : 250) are now put into the culture fluid, to which are then added 

 a few drops of strong sulphuric-acid water (2:1). If any appreci- 

 able quantity of nitrite is present the culture immediately becomes 

 blue-black from the liberation of free iodine, which acts upon the 

 starch. Old potassium iodide water should never be used without 

 first testing carefully, as it usually contains some free iodine. It 

 is always best to first make a trial test without the bacteria. Com- 

 mercial starch frequently contains products of bacterial decomposi- 

 tion and starch prepared aseptically should be substituted. 



A test for ammonia gas. For this Nessler's reagent may be 

 used. 



Nessler's reagent. Dissolve 50 grams potassium iodide in a 

 minimum quantity of cold water. Add a saturated solution of mer- 

 curic chloride until a slight but permanent precipitate persists. 

 Add 4oO cc. of 50 per cent solution of potassium hydrate, made by 

 dissolving the potassium hydrate and allowing it to clarify by sedi- 

 mentation before using. Dilute to one liter, allow to settle ana 

 decant. This solution should give the required color with ammonia 

 within five minutes after addition, and should not precipitate with 

 small amounts of ammonia within two hours. 



