50 CULTURE MEDIA. 



of alcohol, should be added to one cubic centimetre of bouillon. The 

 beginning of an alkaline reaction is indicated by the appearance of a 

 faint rose color. Fuller (1895), who has made a careful investiga- 

 tion of this subject, recommends a modification of the method of 

 Schultz. He gives the following directions in his paper published 

 in the Journal of the American Public Health Association (Vol. 

 XX., p. 386): 



This indicator is prepared by dissolving- five grammes of commercial 

 phenolphthalein in one litre of fifty-per-cent alcohol. It is not feasible to use 

 this indicator 011 strips of paper as the alcohol quickly evaporates, leaving the 

 powder. The colorless liquid, however, may be added in small quantities to 

 solutions of an acid or neutral nature without any change of color, but alka- 

 lies quickly change it to a purple red. This change from 110 color to one of 

 purple red makes the indicator a very satisfactory one, owing to the ease 

 with which the eye detects the so-called end- point. 



For the determination of the degree of reaction of nutrient media it is 

 the custom to put five cubic centimetres (practically five drachms) of the solu- 

 tion into a six-inch porcelain evaporating dish, together with forty-five cubic 

 centimetres of distilled water. This liquid is boiled for three minutes, after 

 which is added one cubic centimetre of the phenolphthalein solution. While 

 the solution is still hot it is quickly titrated against a twentieth normal solu- 

 tion of caustic alkali. 



As already mentioned, Dahmen stated that this indicator is useless, ow- 

 ing to the presence of carbonates, and of ammonia and its salts. On gen- 

 eral grounds the point that this indicator is inaccurate under such conditions 

 is well taken, but so far as its application to nutrient media is concerned it 

 has been found that by proper precautions these objections may be over- 

 come. 



With regard to the amount of free and combined ammonia present in 

 ordinary nutrient media at the times when their reaction is determined, it 

 has been found that it does not exceed 0.003 per cent. Experiments show 

 that this quantity is less than one- tenth of that necessary to interfere with 

 the accuracy of the method. It may be added that the reason why no am- 

 monia is produced by the addition of alkali to the nitrogenous bodies is that 

 at no time during the preparation is there an appreciable excess of free alkali 

 present. 



The chief point by which the presence of carbon dioxide is obviated is by 

 the use of caustic soda instead of sodium carbonate for neutralization, as re- 

 ferred to beyond. It has been learned by actual experiment that the carbon 

 dioxide is practically all removed by heat during the preparation of the 

 media and at the time of boiling just prior to the titration. In order to pre- 

 vent atmospheric absorption of this gas the titration should be made quickly 

 and in a hot solution. 



The remaining precaution concerns the solution against which the nu- 

 trient media are titrated. All of the usual media react acid to phenolphtha- 

 lein ; hence the solution in question must be alkaline. Caustic soda serves 

 the purpose well, and the strength may be conveniently one-twentieth normal, 

 equal to two grammes per litre. For the sake of prevention of interference 

 from carbon dioxide in the standard solution as it meets the indicator, it is 

 well to add a small quantity of calcium hydrate in order to precipitate this 

 gas as calcium carbonate and allow it to settle out in the reagent bottle. 

 After this solution has been accurately prepared, care is necessary in order 

 to keep it of uniform strength and free from carbon dioxide. This is best 

 done by placing the stock solution bottle on a shelf from which the liquid 

 may be delivered into the burette by means of a siphon that is connected 

 tightly with the top of the burette. In the tightly fitting stopper of the bottle 



