CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



529 



blood slowly and stir to avoid the 

 formation of clumps. 



(5) Boil in the steamer for 40 minutes. 



(6) Filter (method not given). 



(7) Sterilize (method not given). 

 (The agar is light red. ) 



(8) To a liter of agar add 300.0 cc. of 

 infusion broth or 1.0 to 1.5% 

 Liebig's extract broth, 4.0 g. lac- 

 tose, 4,0 cc. alcoholic concentrated 

 fuchsin solution and 2.0 cc. of a 

 10% NazSOs solution. (Alkali 

 need not be added.) 



(c) Zipfel prepared regenerated Endo 

 agar as follows: 



(1) Remove the Endo agar slants and 

 plates from tubes and petri dishes. 



(2) Add 3 0% HCl to used Endo agar 

 slants or plates until the HCl 

 covers the agar. Stir. 



(3) Allow the acid to react for one 

 hour, stirring continuously. 



(4) Pour the red agar on a sieve. The 

 HCl solution may be saved and 

 utilized (see end). 



(5) Wash the pieces of agar with a 

 stream of water until the wash 

 water is clear. 



(6) Soak the agar in water for 24 hours, 

 changing the water frequently. 



(7) Place the agar on a sieve at the 

 end of this time and allow to drip 

 free from water. 



(8) Liquify the agar particles in 

 streaming steam. 



(9) To a liter of (8) add 8 to 10.0 cc. 

 of a 10% soda solution, 4.0 cc. of 

 a filtered and sterilized solution of 

 20.0% peptone and 20.0% meat 

 extract or meat equivalent. 



(10) Sterilize for one hour in the 

 steamer. 



(11) To prepare Endo agar add 3.0 cc. 

 of a concentrated fuchsin solution, 

 25.0 cc. of a 10.0% NajSOs solution 

 freshly prepared and 8.0 g lactose 

 dissolved in a little water, per 

 liter of (10). 



(12) Pour into sterile plates. 



(13) To utilize the HCl washings from 

 (4), boil and then neutralize with 

 soda, filter (charcoal may be 

 added) and concentrate by evap- 

 oration. The washings may be 



used directly in the preparation of 

 agar after neutralization and fil- 

 tration by adding 2 or 3% peptone 

 per liter. 

 (14) To prepare plain agar from used 

 Endo agar take the slants and 

 plates and treat with a 0.5% NaOH 

 solution after washing (5) until the 

 red color disappears. Wash in 

 water until the agar reacts neutral. 

 Add peptone and meat extract as 

 in (9). To completely remove the 

 pink color add 1.0 cc. of a 1.0% 

 bismark brown solution per liter 

 of agar, 

 (d) Levine gave the following method of 

 preparation as Kendal's modification : 



(1) Prepare plain, sugar-free nutrient 

 agar, using 15 grams of agar per 

 liter. 



(2) Adjust the reaction to a point just 

 alkaline to litmus. 



(3) Flask the agar, 100.0 cc. to a flask, 

 and sterilize in the autoclave. 



(4) Prepare a 10.0% solution of basic 

 fuchsin in 96.0% alcohol. This 

 solution is fairly stable if kept 

 away from light. 



(5) Prepare a 10.0% aqueous solution 

 of chemically pure anhydrous 

 sodium sulphite (1.0 g. in 10.0 cc. 

 water). This solution does not 

 keep. 



(6) Add 1.0 cc. of (4) to 10.0 cc. of (5) 

 and heat in the Arnold sterilizer 

 for 20 minutes. The color of the 

 fuchsin is nearly discharged if the 

 solutions are of proper strength. 

 This solution must be prepared 

 each day — it does not keep. 



(7) Add 1.0 g. of C. P. lactose (free 

 from glucose) to 100.0 cc. of agar 

 and place in the autoclave until 

 melted and the lactose is thoroly 

 dissolved. 



(8) Add a sufficient volume of (6) 

 (about 1.0 cc.) to impart a faint 

 pink color to the medium. 



(9) Pour into sterile Petri dishes and 

 allow to harden in a dark place 

 with the covers partly removed. 

 When cool the medium should be 

 colorless when viewed from above 

 and a very faint pink when viewed 



