34 METHODS OF CULTIVATION OF BACTEEIA 



sufficiently got over by making up a solution of disodic 

 phosphate (Na 2 HP0 4 ,2H 2 0), 1T876 grm. to the litre; test 

 paper immersed in this assumes a tint just on the alkaline 

 side of what is usually regarded as the optimum reaction for 

 bacterial growth. In applying this method it is preferable to 

 use azolitmin papers (made by immersing filter paper in O'l 

 per cent, of the dye overnight and drying) or neutral-red papers 

 (made by treating the paper with 0'02 per cent, neutral-red for 

 three minutes). Both of these papers are more sensitive than 

 ordinary litmus paper. 



Eyre's Method. — Several methods have been introduced for 

 adjusting the reaction by titration, and that of Eyre is widely 

 used. It is applicable to any of the media ordinarily employed. 



Preparation of Standard Solutions. — The first requisites here are 

 normal solutions of aeid and alkali. The latter is prepared as follows : 

 85 grammes of pure sodium bicarbonate are heated to dull redness for 

 ten minutes in a platinum vessel and allowed, to cool in an exsiccator. 

 Just over 54 grammes of sodium carbonate should now be present ; any 

 excess is quickly removed, and the rest being dissolved in one litre of 

 distilled water, a normal solution is obtained. A measured quantity is 

 placed in a porcelain dish, and a few drops of a '5 per cent, solution 

 of phenol-phthalein in neutral methylated spirit is added to act as 

 indicator. The alkali produces in the latter a brilliant rose-pink, which, 

 however, disappears on the least excess of acid beiDg present. The 

 mixture is boiled and a solution of hydrochloric acid of unknown strength 

 is run into the dish from a burette till the colour goes and does not 

 return after very thorough stirring. The strength of the acid can then 

 be calculated, and a normal solution can be obtained. From these two 

 solutions any strength of acid or alkali (such as the decinormal solution 

 of NaOH mentioned below) may be derived. The soda solutions are best 

 stored in bottles with such a cork as is shown in Fig. 38 ; on the air 

 inlet is placed a little bottle filled with soda lime and fitted with a 

 similarly tubed cork. The C0 2 of the air which passes through is thus 

 removed. 



As Eyre has suggested, the reaction of a medium may be 

 conveniently expressed by the sign + or - to indicate acid or 

 alkaline respectively, and a number to indicate the number of 

 cubic centimetres of normal alkaline or acid solution necessary 

 to make a litre of the medium neutral to phenol-phthalein. 

 Thus, for example, " reaction = - 15," will mean that the 

 medium is alkaline, and requires 15 c.c. of normal HC1 to make 

 a litre neutral. It has been found that when a medium such 

 as bouillon reacts neutral to litmus, its reaction to phenol- 

 phthalein, according to the above standard, is on the average 

 + 25. Now, as litmus was originally introduced by Koch, and 

 as nearly all bacterial research has been done with media tested 

 by litmus, it is evidently difficult to say exactly what precise 



