CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



299 



Use : To study bacterial nutrition of Bacil- 

 lus influenzae. Authors reported growth 

 in a blood cell dilution of 1:100,000. 



Reference: Thjotta and Avery (1921 

 p. 109). 



976. Thjotta and Avery's Yeast Hemoglobin 

 Bouillon 



Constituents : 



1. Distilled water 410.0 cc. 



2. Brewer's yeast 100.0 g. 



3. Bouillon 



4. Hemoglobin (crystalline) .... 1.0 g. 

 Preparation : 



(1) E.xact method of preparation of 

 bouillon not given. 



(2) Adjust the reaction of (1) to pH 

 = 7.8. 



(3) Emulsify 100.0 g. of brewer's yeast 

 in 400.0 cc. distilled water with the 

 reaction of pH= 4.6. 



(4) Boil over a free flame for 10 minutes. 



(5) Allow to sediment at room tem- 

 perature. 



(6) Pipette off the clear supernatant 

 extract and test sterility. 



(7) Store in the ice chest until ready for 

 use. Readjust the reaction of the 

 extract from pH = 7.3 to 7.5 just 

 before use. 



(8) Prepare crystalline hemoglobin by 

 the method of Walker and William- 

 son. J. Biol. Chem., 41: 75, 1920. 



(9) Prepare a 10.0% solution of crys- 

 talline hemoglobin in water by 

 weight. 



(10))Dilute sterile (9) with sterile bouil- 

 lon in varying proportions from 

 1:10 to 1:200,000. 

 (11) Add yeast extract (7) amount not 

 given. 



Sterilization: Method of sterilization of 



I, bouillon not given. Sterilization of yeast 

 infusion effected by boiling in step (4). 

 Filter the hemoglobin solution thru a 

 Berkefeld filter to sterilize. 



Use: To study bacterial nutrition of Bacil- 

 lus influenzae. Authors reported no 

 growth of B. influenzae without yeast 

 extract. Growth when hemoglobin solu- 

 tion diluted 1:200,000 (with yeast extract 

 present). 



Reference: Thjotta and Avery (1921 

 p. 109). 



977. Savini and Savini-Castano's Bacteria 

 Blood Bouillon 



Constituents: 



1. Bouillon. 



2. Glycerol. 



3. Blood. 



4. Staphylococcus aureus. 

 Preparation : 



(1) Place 5.0 cc. of glycerol in a small 

 Erlenmeyer flask containing glass 

 beads. 



(2) Wash the growth from 2 or 3 cultures 

 of Staphylococcus aureus (24 to 48 

 hours cultures at 37°C.) and place in 

 sterile (1). (Bouillon cultures may 

 be used.) 



(3) Place in a paraffin oven at 58 to GO'C. 

 over night until the bacteria are 

 kUled. 



(4) Add 3.0 to 4.0 cc. of blood freshly 

 drawn under aseptic conditions to (3). 



(5) Shake until the blood is coagulated 

 completely. 



(6) Heat at 58 to 60°C. for about an hour. 

 (When heating here and in step (3) 

 place the cotton stopper in the tube 

 or flask lightly to allow evaporation 

 of water.) 



(7) Add sufficient of (6) to sterile bouillon 

 until the bouillon takes on a weak 

 red color. 



Sterilization: Method of sterilization of 

 glycerol not specified. Method of steril- 

 ization of bouillon not given. 



Use: Cultivation of influenza bacilli. 



Variants: The author prepared a similar 

 medium using a hemoglobin solution in- 

 stead of blood. The powdered hemo- 

 globin was sterilized at 100°C. in the hot 

 air sterilizer after drying in the desic- 

 cator. The powdered hemoglobin was 

 crushed in a sterile mortar with solution 

 obtained in step (3) above. Filter thru 

 sterile filter paper. This solution was 

 used instead of blood as indicated in 

 step (6) and (7). 



Reference: Savini and Savini-Castano 

 (1911 p. 494). 



SUBGROUP I-C. SECTION 14 



Liquid media or basal solutions contain- 

 ing a digest or autolysate other than a com- 

 mercial digest. 



