CULTIVATION OF BACTERIA 53 



of normal soda solution can be added for each litre of neutral media; 

 if an acidity is desired, normal hydrochloric acid solution is added. 



Many bacteriologists consider that litmus is not delicate enough to 

 be entirely satisfactory, especially when experiments are to be reported 

 or exactly repeated. This objection is made chiefly by those investi- 

 gating water bacteria who are watching cultural and biochemical charac- 

 teristics in simple peptone-beef media. For these purposes phenol- 

 phthalein has been generally selected. It is of great importance to re- 

 member that different indicators not only differ in delicacy, but that they 

 react differently to different substances. A medium which is alkaline to 

 litmus is acid to phenolphthalein, showing that there are present in such 

 media substances possessing an acid character which litmus does not 

 detect. These substances are weak organic acids and organic com- 

 pounds, theoretically amphoteric, but in which an acid character pre- 

 dominates. Thus, a litre of bouillon becomes, on the addition of 1 per 

 cent, of peptone, more alkaline to litmus, but decidedly more acid to 

 phenolphthalein; 100 c.c. of water with 1 per cent, of peptone is acid 

 to phenolphthalein to such an extent that 3.5 c.c. of decinormal NaOH 

 is required to neutralize it. To litmus it is alkaline and requires 3.4 c.c. 

 of decinormal HC1. Two per cent, of peptone doubles the difference. 

 The same figures hold approximately true for peptone broth. We 

 should find by growing the bacteria just what reaction we want for any 

 variety, and then test the fluid with phenolphthalein or litmus as the 

 indicator. With precisely similar ingredients we can then exactly re- 

 produce at any time in the future the same reaction, but with different 

 materials one would again have to study the reaction. 



Titration of Culture Media. We must have accurately standard- 

 ized normal and decinormal solutions of sodium hydrate and hydro- 

 chloric acid ; also a 0.5 per cent, solution of phenolphthalein in 30 per 

 cent, alcohol and a neutral 1 per cent, solution of Merck's litmus. 



Care should be taken to prevent the absorption of carbon dioxide by 

 the soda solution, by arranging that all air which comes in contact with 

 the latter, either in the stock bottle or in the burette, shall first pass 

 through a strong solution of sodium or barium hydrate. The arrange- 

 ment of the apparatus is described in any work on chemical analysis. 

 The medium is brought to the desired volume with w r ater, and boiled 

 four minutes to expel the carbon dioxide. Media are commonly warm or 

 hot when measured, hence it must be remembered that true volumes 

 cannot be thus obtained ; for instance, a litre measured at, say, 80 C. 

 would be only 973 c.c. if measured at 20 C., the temperature at which 

 litre flasks are calibrated. Since many media cannot be cooled to 20 C. 

 because of solidification, as in the case of agar or gelatin, it is a better 

 plan when accuracy is important to determine measures of volume by 

 weight. For this, place a clean, dry saucepan, in which the medium is 

 to be prepared, upon one side of a trip scale, and counterbalance its 

 weight exactly. The weight of a litre of bouillon, gelatin, or agar 

 having been determined once for all, the necessary weights added to 

 the weight of the pan will give the amount which the pan and its con- 



