Cultivation of Bacteria 191 



prescribed volume by the addition of distilled water to replace that lost by boiling, 

 and after being thoroughly stirred, S cc. are transferred to a 6-inch porcelain 

 evaporating-dish. To tJiis 45 cc. of distilled water are added and the 50 cc. of 

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

 solution of phenolphthalein (No. i) 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 



20 



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 developed, 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 addi- 

 tion 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 reaction always to be obtained. All titra- 

 tions 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 calcu- 

 lated 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 thoroughly 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 i 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 addition of sodium hydroxid, and the titrations and additions of alkali 

 must be repeated until the mediuni has the desired reaction {i.e., 0.0 per cent, to 

 0.005 per cent.; see below). 



"After the prescribed period of heating, it is frequently found that the medium 

 is again slightly acid, usually about 0.5 per cent. Without correcting this, the 

 fluid is to be filtered and the calculated amount of acid or alkali is to be added 

 to change the reaction to the one desired. A still further change in reaction is 

 not infrequently to be observed after sterilization, the degree of acidity varying 

 apparently with the composition of the media and the degree and continuance 

 of the heat." 



"Manner of expressing the reaction: Since at- the time the reaction is first 

 determined culture-media are more often acid than alkaline, it is proposed that 

 acid media be designated by the plus sign and alkaline media by the minus sign, 

 and that the degree of acidity or alkalinity be noted in parts per hundred. Thus, 

 a medium marked -f 1.5 would indicate that the medium was acid, and that 1.5 



per cent, of - sodium hydroxid is required to make it neutral to phenolphthalein; 



while —i.s would indicate that the medium was alkaline and that 1.5 per cent. 



of - acid must be added to make it neutral to the indicator." 



"Standard reaction of media (provisional): 



''Experience seems to vary somewhat as to the optimum degree of reaction 

 which shall be uniformly adopted in the preparation of standard culture-media. 

 To what extent this is due to variation in natural conditions as compared with 

 variations of laboratory procedure it seems impossible to state. Somewhat 

 different degrees of reaction for optimum growth are required, not only in or 

 upon the media of different composition and by bacteria of different species, but 

 also by bacteria of the same species when in different stages of vitality. ' The 

 bulk of available evidence from both Europe and America points to a reaction 

 of -f I.s as the optimum degree of reaction for bacterial development in inoculated 

 culture media. While this experience is at variance with that in several of our 

 own laboratories, it has been deemed wisest to adopt +1.5 as the, provisional 



