22 CULTURE MEDIA 



the addition of a drop fails to show any intensifying of the purplish-violet color at the 

 spot where it came in contact with the diluted bouillon in the beaker. This marks 

 the end reaction. A reaction of about +0.7 in the cold gives a delicate pink with 

 phenolphthalein as an indicator. Titration in the cold is not very satisfactory with 

 gelatin and agar. 



Having determined the percentage acidity of the 10 c.c. sample tested, we easily 

 calculate the number of c.c of N/i NaOH solution required to be added to the 1000 

 c.c. of bouillon to obtain a reaction corresponding to the neutral point of phenol- 

 phthalein. It is more exact to take the average of two titrations. 



As TOO c.c. of medium would require 3^ c.c., 1000 c.c. would require 10 times as 

 much, or 35 c.c. N/i NaOH solution. Having measured out and added 35 c.c. 

 of the N/i NaOH solution to the meat infusion, containing salt and peptone, we 

 have a solution which is exactly neutral to phenolphthalein, or o. It is usually con- 

 sidered that a reaction of about i% acid is the optimum reaction for bacterial 

 growth. Hence we should now add i% of N/i HC1 solution to the medium. This 

 would be accomplished by adding 10 c.c. of N/i HC1 solution to the 1000 c.c. of 

 neutralized medium, and we would have a medium with a reaction of -f-i. If we 

 desired a reaction of i% alkalinity we would add an additional c.c. of N/i NaOH 

 solution to every 100 c.c. of the medium at o, or 10 c.c. for the 1000 c.c. of medium. 

 The reaction would then be i. 



As a matter of convenience, we usually determine the reaction of the 

 medium, which is always more or less acid, and then add enough N/i 

 NaOH to reduce the acidity to the percentage we desire to set the med- 

 ium, instead of neutralizing all the acidity present and then, in a second 

 operation, restoring the acidity to the point desired. 



Thus finding the acidity of the medium to be 3^% and desiring to give it an 

 acidity of i%, we would add only 2% c.c. of N/i NaOH to every 100 c.c. of medium, 

 or 25 c.c. for the 1000 c.c. of medium. The reaction would then be found to 

 be + i. 



The neutral point of litmus is not a sharp one, but it corresponds rather closely 

 with a reaction of +1.5 to phenolphthalein. The recommendations of the A. P. H. 

 Association call for making the titration with the medium boiling. If the color of the 

 end reaction at boiling-point be obtained, it will be found that when cool it deepens 

 until it corresponds to the rich violet-pink of the end reaction in the cold or vice versa. 



To summarize: 



Take Peptone 10 grams 



Sodium chloride. 5 grams 



50% meat infusion 1000 c.c. 



Dissolve the peptone and sodium chloride in the meat infusion and 

 add enough N/i NaOH to make the reaction +i. 



Put the solution in the inner compartment of a rice cooker and bring to the boiling- 

 point and maintain this temperature for twenty minutes. The calcium chloride or 



