574 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



(13) Remove blood aseptically with a 

 syringe from the median basilic or 

 median cephalic veins of patient 

 (15.0 cc). 



(14) Divide the 15.0 cc. blood among eight 

 sterile melted tubes of (11), 2.0 cc. 

 per tube. 



(15) Add ascitic fluid to each tube of 

 (13), i to J volume (i.e., 6-10.0 cc.) 

 ascitic fluid must be clear, free from 

 bile or blood pigment and have a 

 specific gravity of more than 1.015. 

 Must not be filtered, sterilized or 

 contain preservatives. 



(16) Mix the content of each tube thoroly 

 by pouring once or twice into 

 another sterile tube. Avoid air 

 bubbles. 



(17) After thoro solidification cover each 

 one of the (15) with 2 or 3 cm. of 

 sterile melted agar (11). 



(18) To prevent drying, paraffin the 

 cotton stopper of each tube. 



References: Plotz, Olitsky and Baehr 

 (1915 p. 6), Baehr and Plotz (1917 p. 203). 



1839. Reed and Orr's Blood Agar 



Constituents : 



1. Water 1000.0 cc. 



2. Peptone 5.0 g. 



3. Agar 15.0 g. 



4. Beef extract 3.0 g. 



5. Rabbits blood (whole) 10.0 cc. 



6. Phosphates to 0.05M 



Preparation : 



(1) Prepare beef extract peptone agar 

 according to Committee A. P. H. A. 

 (1916-1917). (See medium 1695.) 



(2) Adjust with NaOH to pH = 7.4. 



(3) Add phosphate mixture of pH = 

 7.4 to make finished medium 0.05M 

 phosphate. 



(4) Add whole rabbits blood to sterile 

 (3) cooled to 80°C. to make finished 

 medium 1.0% blood. 



(5) Maintain at 80°C. for 10 minutes. 



(6) Tube or plate. 



Sterilization: Sterilize in the autoclave. 



Use: To maintain cultures of Hemophilus 

 influenzae. Author reported that pH 

 range of 6.8 to 7.4 gave very nearly 

 maximum growth. Marked morphologi- 



cal changes did not appear except near 

 the limiting pH values. 

 Reference: Reed and Orr (1923 p. 104). 



1840. Becker's Defibrinated Blood Agar 



Constituents : 



1. Extract agar 1000.0 cc. 



2. Blood, defibrinated 175.0 cc. 



Preparation : 



(1) Prepare extract agar according to 

 Standard Methods, see medium 1695, 

 and distribute in 40.0 cc. lots in 100.0 

 cc. flasks. 



(2) Melt agar and cool to 45-50 °C. 



(3) Add 1.0 cc. of defibrinated human 

 blood per 6.0 cc. agar. 



(4) Mix thoroly. 

 Sterilization: Method not specified. 



Use: To isolate streptococci, pneumococci 



and gonococci. 

 Reference: Becker (1916 p. 759), Tanner 



(1919 p. 69). 



1841. Esch's Alkaline Hemoglobin Ragit 

 Agar 



Constituents : 



1. Distilled water 7.5 cc. 



2. NaOH (normal 7.5 cc. 



3. Hemoglobin (Merck) 2.5 g. 



4. Ragit agar 85.0 cc. 



Preparation : 



(1) Grind 2.5 g. Merck's hemoglobin to a 

 powder in a mortar. 



(2) Place in a balloon flask and add 7.5 

 cc. of N/1 caustic soda solution and 

 7.5 cc. of distilled water. 



(3) Heat by means of a gas burner and 

 stir strongly to obtain complete 

 solution of the hemoglobin which 

 requires about 10 minutes. 



(4) Heat (3) for one hour in a Koch 

 steamer, or for 15 minutes in an auto- 

 clave at one atmosphere pressure. 



(5) Prepare "Ragit agar." 



(6) Melt sterile (5) and add (4) to 85.0 

 cc. of the agar. 



(7) Mix well and distribute into sterile 

 petri dishes. 



Sterilization : Step (4) above gives steriliza- 

 tion of alkaline hemoglobin. Method of 

 sterilization of Ragit agar not given. 



