TITRATION AND ADJUSTMENT OF CULTURE MEDIA 225 



40° and 50°C. was used. This was reduced later to about 30°C.^' 

 Five cubic centimeters of the hot agar are added by means of 

 a pipet to 45 cc. of distilled water — temperature about 30°C. 

 (verified with a thermometer) . One cubic centimeter of phenol- 

 phthalein (1 per cent solution in 50 per cent alcohol) is added 

 and the titration performed at once. 



It is now our custom to titrate each batch of neutral veal 

 agar at least twice during its preparation making the necessary- 

 adjustments of reaction. Here, as with the broth, allowance 

 must be made for further heating in the autoclave. Experience 

 shows that agar made from meat infusion rises in acidity usually 

 about 0.3 to 0.4 per cent, at 15 pounds pressure during one and 

 one-half to two hours. ^^ Therefore, 3 to 4 cc. of normal soda 

 should be added per liter in excess of the amount required to 

 secure the phenolphthalein neutral point at the time of the 

 first titration. The second titration is made just after filtra- 

 tion, before tubing and sterilizing. If the amount of soda needed 

 does not exceed 0.2 per cent, little if any precipitate occurs 

 on heating further. If more than the above amount is needed 

 in adjusting the reaction, the medium should be heated in the 

 Arnold for half an hour and the precipitate filtered out, before 

 tubing and sterilizing. 



In very careful work the medium is also titrated a third time 

 as it comes from the autoclave. For the last test a tube or small 

 bottle of neutralized glassware should be used in order that the 

 reaction of the agar may be unaffected by its container. This 

 sample is tested before it hardens — as remelting would raise its 

 acidity further. On the addition of phenolphthalein, it should 

 show a very dehcate shade of pink if it is "neutral." 



REMELTING OF SOLID MEDIA 



An important factor to be considered in the adjustment of 

 media is the remelting of sohd media for the addition of sterile 

 substances such as blood, serum, etc., or for the purpose of 



1^ This slight difference of temperature had no noticeable effect on the results 

 of the titration. 



18 This time is necessary for melting (and clearing with egg) of large batches 

 of agar (5 to 12 liters). 



