34 METHODS OF CULTIVATION OF BACTEEIA 



neutral. It has been found that when a medium such as bouillon 

 reacts neutral to litmus, its reaction to phenol-phthaleine, accord- 

 ing 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 degree of 

 alkalinity is the optimum for bacterial growth. It is probably 

 safe to say, however, that when a medium has been rendered 

 neutral to phenol-phthaleine by the addition of NaOH, the 

 optimum degree is generally attained by the addition of from 

 10 to 15 c.c. of normal HC1 per litre, i.e. the optimum reaction 

 is + 10 to + 15. In other words, the optimum reaction for 

 bacterial growth lies, as Fuller has pointed out, about midway 

 between the neutral point indicated by phenol-phthaleine and 

 the neutral point indicated by litmus. 



The only objection to the use of phenol-phthaleine is that its 

 action is somewhat vitiated if free CO 2 be present. This can 

 be completely obviated as follows. Before testing any medium 

 it is boiled in the porcelain dish into which titration takes place. 

 The soda solutions are best stored in bottles such as that shown 

 in Fig. 44, having on the air inlet a little bottle filled with soda 

 lime with tubes fitted as in the large one. The C0 2 of the air 

 which passes through is thus removed. 



Method. The following procedure includes most of the 

 improvements introduced by Eyre. The medium with all its 

 constituents dissolved is filtered and then heated for about 45 

 minutes in the steamer, the maximum acidity being reached 

 after this time. Of the warm medium take 25 c.c. and put in 

 a porcelain dish, add 25 c.c. distilled water, and 1 c.c. phenol- 

 phthaleine solution. Run in decinormal soda till neutral point 

 is reached, indicated by the first trace of pink colour, the mixture 

 being kept hot. 1 Repeat process thrice, and take the mean ; 

 this divided by 10 will give the amount (x) of normal soda 

 required to neutralise 25 c.c. of medium ; then 40 x = amount 

 necessary to neutralise a litre; and 40 #-10 = amount of 

 normal soda necessary to give a litre its optimum reaction. 

 Then measure the amount of medium to be dealt with, and add 

 the requisite amount of soda solution. 



1 The beginner may find considerable difficulty in recognising the first 

 tint of pink in the yellow bouillon. . A good way of getting over this is to 

 take two samples of the medium, adding the indicator to one only ; then to 

 run the soda into these from separate burettes ; for each few drops run into 

 the medium containing the indicator the same amount is run into the other. 

 Thus the recognition of the first permanent change in tint will be at once 

 recognised by comparing the two lots of solution. 



