THE PREPARATION OF CULTURE MEDIA 153 



titrated and sufficient alkali added to make the reaction about two or three 

 points more alkaline than the desired final reaction of the medium. 



Solution (2) to 500 c.c. of water add the quantity of agar that will 

 give the right concentration for the final volume (1 liter). For instance, 

 if a 3 per cent agar is desired, add 30 grams of agar to 500 c.c. of water 

 and dissolve in the autoclave at fifteen pounds pressure for fifteen minutes. 

 When the agar is dissolved, cool it down to 50 C. and add to it Solution (1). 

 Mix thoroughly and titrate again. Usually the addition of the agar solution 

 does not change the reaction. Add the whites of two eggs well beaten with 

 a little water and autoclave for thirty minutes at fifteen pounds pressure. 

 If no autoclave is available, the medium may be put in the Arnold for 

 forty-five minutes. The autoclave, however, is more satisfactory. It is best 

 to check the titration again after autoclaving. 



The medium is now filtered, tubed and sterilized. 



LACTOSE-LITMUS- AGAR (Wurtz). This is ordinary meat extract agar 

 which is adjusted to P^ 7.5 to 7.8, to which 1 per cent of lactose is added, 

 and enough litmus to give it a bluish purple color when cooled. Instead 

 of litmus, 1 per cent of the Andrade indicator can be used. If the latter 

 indicator is used, the reaction of the medium must be brought down to P^ 

 7.2. The medium containing 1 per cent of the indicator, if the reaction is 

 correct, will be dark red when hot and colorless when cold. After the 

 addition of the sugar and the indicator, it is best to sterilize by the inter- 

 mittent method on three successive days. 



The red color fades out gradually, the indicator should be yellow after 

 standing 2 or 3 hours. If it remains red or reddish, 1 or 2 c.c. more of 

 normal NaOH should be added. 



The above description of the basic media, broth and agar, details chiefly 

 the methods in general use until a few years ago. The recognition that 

 bacteria grow more luxuriantly upon media containing considerable quantities 

 of protein-split products, has resulted in the utilization of trypsinized culture 

 media which has been found to possess considerable advantages for the 

 cultivation of delicate organisms like the meningococci, etc. A simple formula 

 for the production of trypsinized agar is the following, taken from the 

 directions of Gordon : 17 



TRYPAGAR. To one pound of chopped meat, free from fat, add 1 liter 

 tap water and make faintly alkalin to litmus with 20 per cent NaOH 

 solution. Heat in double boiler at 75 to 80 for five minutes. Cool to 37 

 and add 0.5 gram trypsin. (Fairchild's preparation. For other brands the 

 amount must be determined by experiment.) Incubate for five to six hours. 

 Test for pepton as follows: Take 5 c.c. of the liquid, add 5 c.c. N/l NaOH 

 and 1 c.c. dilute CuSO 4 . A pink color indicates that trypsinization is 



17 Gordon, Br. Med. Journal 2, 1916, 678. 



