CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



585 



(14) Sterilize in an autoclave at 15 



pounds for 30 minutes. This 



medium was recommended by the 



authors to be used in Avery's 



Oleate Blood Cell Agar, see 



medium 1858. 



References: Huntoon (1918 pp. 170, 172), 



Harvey (1921-22 p. 120), Torrey and 



Buckeil (1922 p. 127), Park, Williams and 



Krumwiede (1924 p. 126). 



1864. Harvey's Alkaline Egg Agar 



Constituents: 



1. Water 500.0 cc. 



2. Whites of 2 eggs 



3. Yolk of 1 egg 



4. N/1 NaOH 6.0 cc. 



5. Infusion agar 5000.0 cc. 



Preparation : 



(1) Mix the whites of two eggs, the yolk 

 of one egg and 6.0 cc. of normal 

 NaOH. 



(2) Add 500 .p cc. of water. 



(3) Heat very slowly to 95°C. 



(4) Keep at 95°C. for 60 minutes. 



(5) Filter thru cotton wool. 



(6) Distribute into flasks. 



(7) When ready for use mix 10 parts 

 sterile melted infusion agar, cooled to 

 45''C. (see variant (v) medium 1661 

 for preparation) with one part sterile 

 (6) heated at 45°C. 



Sterilization: Sterilize (6) in the autoclave. 

 Use: General culture medium. 

 Variants : 



(a) Harvey reported that a clearer 

 medium was obtained if the whites 

 of two eggs were mixed with 4.0 cc. 

 of normal NaOH and 330.0 cc. of 

 water instead of the quantities given 

 above. 



(b) Harvey prepared a similar medium as 

 follows: 



(1) Mix the whites of two eggs, yolk of 

 one egg and 6.0 cc. of normal NaOH. 



(2) Add 500.0 cc. of water. 



(3) Heat very slowly to 90°C. 



(4) Distribute into flasks. 



(5) Sterilize (4) in the autoclave. 



(6) When ready for use, mix one part 

 (5) heated to 45°C. and ten parts 

 sterile melted infusion agar cooled 

 to 45°C. (see variant (v) medium 

 1661 for preparation). 



Reference: Harvey (1921-22 p. 86). 



1865. Meyer's Basal MoUusk Infusion Agar 

 Constituents: 



1. Tap Water 1000.0 cc. 



2. MoUusks 500.0 g. 



3. Peptone 10.0 g. 



4. Agar 20.0 g. 



5. Egg 2 



Preparation : 



(1) Place 500.0 g. of finely chopped mol- 

 lusks (or beef heart), two whole eggs 

 and 1000.0 cc. of tap water in a 

 double boiler over a free flame and 

 infuse at 60°C. with constant stirring 

 f,or 5 minutes. 



(2) Add 20.0 g. of flaked agar and 10.0 g. 

 peptone (if desired) and raise the 

 temperature until the mixture assumes 

 a brownish color. 



(3) Make slightly alkaline, using a 10.0% 

 Na2C0j solution. 



(4) Heat in the steam sterilizer for one 

 hour. 



(5) Separate the clot from the sides of 

 the container and place in the steri- 

 lizer for another hour. 



(6) Clarify by straining thru glass wool. 



(7) Adjust to pH = 7.2. 



(8) Tube or flask. 



Sterilization: Sterilize the fractional 

 method at 100°C. in flowing steam. 



Use: Cultivation of Cyclostoma elegans 

 and other organisms living in symbiosis 

 with moUusks. 



Added nutrients: 



(a) The author used the basal medium 

 without additional material. 



(b) The author added 10% defibrinated 

 rabbit blood to the melted agar just 

 before use. 



(c) The author added 1.0 to 2.0% of 

 filtered Helix pomatia or Cyclostoma 

 infusion prepared as follows: 



(1) Grind 10 to 20 large sterilized 

 Cyclostomas in a sand mortar. 



(2) Mix (1) with an equal weight of 

 Ringer's solution. The weight of 

 (1) to be as weight of tissue and not 

 of sand and tissue. 



(3) Shake for ten minutes. 



(4) Centrifuge at low speed. 



(5) Remove the supernatant fluid and 

 mix with the hemolymph of about 

 20 to 40 moUusKs. 



(6) Pass thru a diatomatous filter, or 

 sterilize by the addition of chloro- 



