CUL TT VA TION OF BA CTERIA . 1 85 



line to litmus, lacmoid, rosolic acid, and methyl orange, but neu- 

 tral to phenolphthalein and turmeric. 



" Studies made at the Lawrence Experiment Station show that 

 this acid salt may be added to culture-media in amounts greatly 

 exceeding those naturally present in the media without producing 

 any apparent influence upon bacterial development. From these 

 facts it is clear that the use of any of the above-mentioned indi- 

 cators, other than phenolphthalien and turmeric, in the presence 

 of this dibasic phosphate, prevents the addition of a sufficient 

 amount of free alkali to effect neutralization, and as the amount 

 of phosphates in media varies considerably, the reaction passes 

 be>-ond accurate control when litmus and other substances of its 

 class are used as indicators." 



" The question of the proper reaction of media for the cultiva- 

 tion of bacteria and the method of obtaining this reaction have 

 been discussed in a valuable paper by Mr. George W. Tuller, pub- 

 lished in the 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, burettes graduated into one-tenth c.cm. and three solutions 

 are required — 



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



cent, alcohol. 



2. A — solution of sodium hydroxid. 



20 



x. A — solution of h\-dric chlorid. 

 20 J 



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 

 burettes 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.cm. are transferred to a 6-inch porcelain evaporating- 

 dish. To this 45 c.cm. of distilled water are added and the 50 c.cm. 

 of fluid are boiled for three minutes over a flame. One cubic 

 centimeter of the solution of phenolphthalein (No. 1) is then 



