226 Cultivation of Micro-organisms 



'Journal of the American Public Health Association,' Oct., 1895, 

 vol. xx, p. 321." 



"Method of determining the degree of reaction of culture media : 

 For this most important part in the preparation of culture media, 

 burets graduated into one-tenth c.c. and three solutions are required 



"1. A 0.5 per cent, solution of commercial phenolphthalein, in 50 

 per cent, alcohol. 



"2. A solution of sodium hydroxid. 



"3. A solution of hydric chlorid. 



"Solutions 2 and 3 must be accurately made and must correspond 

 with the normal solutions soon to be referred to. 



"Solutions of sodium hydroxid are prone to deterioration from the 

 absorption of carbon dioxid and the consequent formation of sodium 

 carbonate. To prevent as much as possible this change, it is well to 

 place in the bottle containing the stock solution a small amount of 

 calcium hydroxid, while the air entering the burets or the supply 

 bottles should be made to pass through a U-tube containing caustic 

 soda, to extract from it the carbon dioxid." 



" The medium to be tested, all ingredients being dissolved, is brought 

 to the prescribed volume by the addition of distilled water to replace 

 that lost by boiling, and after being thoroughly stirred, 5 c.c. are 

 transferred to a 6-inch porcelain evaporating-dish. To this 45 c.c. 

 of distilled water are added and the 50 c.c. of fluid are boiled for three 

 minutes over a flame. One cubic centimeter of the solution of phenol- 

 phthalein (No. 1) is then added, and by titration with the required 

 reagent (No. 2 or No. 3) the reaction is determined. In the majority of 



instances the reaction will be found to be acid, so that the sodium 



hydroxid is the reagent most frequently required. This determination 

 should be made not less than three times and the average of the results 

 obtained taken as the degree of the reaction. 



"One of the most difficult things to determine in this process is 

 exactly when the neutral point is reached as shown by the color de- 

 veloped, and to be able in every instance to obtain the same shade 

 of color. To aid in this regard, it may be remarked that in bright 

 daylight the first change that can be seen on the addition of alkali is 

 a very faint darkening of the fluid, which, on the addition of more 

 alkali, becomes a more evident color and develops into what might 

 be described as an Italian pink. A still further addition of alkali 

 suddenly develops a clear and bright pink color, and this is the reac- 

 tion always to be obtained. All titrations should be made quickly 

 and in the hot solutions to avoid complications arising from the presence 

 of carbon dioxid. 



"The next step in the process is to add to the bulk of the medium 

 the calculated amount of the reagent, either alkali or acid, as may be 

 determined. For the purpose of neutralization a normal solution of 

 sodium hydroxid or of hydric chlorid is used, and after being thor- 

 oughly stirred the fluid thus neutralized is again tested in the same 

 manner as at first, to insure the proper reaction of the medium being 

 attained. When neutralization is to be effected by the addition of 

 an alkali, it not infrequently happens that after the calculated amount 

 of normal solution of sodium hydroxid has been added, the second 

 test will show that the medium is acid to phenolphthalein, to the 

 extent sometimes of 0.5 to 1 per cent. This discrepancy is perhaps 

 due to side reactions which are not understood. The reaction of the 

 medium, however, must be brought to the desired point by the further 



