CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 587 



5 Agar (Flaked) 18-0 g. 1869. Williams-Burdick's Modified Petroff's 



6 Ascitic fluid 500.0 cc. Egg Infusion Agar (Roddy) 



7. Methyl violet 



Preparation: ^°°'"*."!mI'; . 



(1) Place beef heart (preferably directly 1 . Distilled water 

 from slaughter house) and one whole 2. Eggs 



egg in water. 3. Glycerol 



(2) Place (1) in a double boiler over a 4. Veal ... ■■-■■■■■■■ ^^^"^ S- 



free flame and maintain at 60°C. for 5. Gentian violet (1.0% soln.) 1.0 cc. 



5 minutes with constant stirring. 6. NaCl • §■ 



(3) Add 4 and 5 and raise temperature 7. Agar §• 



until the mixture assumes a brown- Preparation: . , • j „„j^, 



. , , (1) Dilute egg whites obtained under 



(4) ld]usi\o slightly alkaline to litmus aseptic conditions with 10 parts 

 using 10.0% solution of Na.COa- distilled water. Shake thoroly 



(5) Place (4) in flask, or preferably in a (2) To clear, pass the Auid thru a thm 

 coffee pot and keat at 100°C. in layer of cotton and then heat to 

 Arnold steam sterilizer for one hour. 100°C. 



(6) Separate clot from sides of the (3) Filter thru paper. _ 

 receptacle and replace in sterilizer (4) Dilute egg yolk, obtained unde 

 for another hour. aseptic conditions wxth 10 parts 



(7) Clear by centrif uging or by straining distilled water. Mix well. 



^ thru a fine wire mesh and then thru (5) Clarify the cloudy emulsion by the 



glass wool. (Do not use cotton addition of norma NaOH. Too 



cloth or any other material with much NaOH - l^armfuL The emul- 



absorbent properties.) A clear ^^ n I'n ^ tl( Nlont 



medium may also be obtained by Usually LO cc. of -^^^J^^^^^^ 



depositing the meat residue on glass required for each 100.0 cc. of the 



wool in a funnel and the fluid portion emulsion 



allowed to percolate thru. (6) Heat to 100°C. and filter 



(8) AdjustreactiontopH = 6.8. (7) ^ov- 500.0 g.o finely chopped lean 



(9) Heat and tube in 10.0 cc. lots. veal with one liter of water contain- 

 (10) Add 5.0 cc. ascitic fluid to each tube ing 15.0% glycerol. 



of sterile melted medium just before (8) Infuse for 24 hours and filter 



(9) Add 5.0 g. NaCl to the filtrate ot 



SterilizTonf Sterilize (9) in autoclave at (8) and heat to boiling. 



12 pounds pressure for 10 minutes but (10) Filter. x , i nor 



preferably at 100°C. in flowing steam (11) Adjust the reaction to +1.0% 



using the fractional method. alkaline. r+ . flo=t- 



Use: Isolation of gonococcus. Authors (12) Place 300.0 cc. of (3) m a liter flask 



reported that colonies after 24 hours were (13) Place 300.0 cc. of (6) m another 



about 1.0 mm. in diameter. May show a flask. j ir n ^ ..f 



smoky tinge by transmitted light. May (14) Place 400.0 cc. of 11) and 15.0 g. of 



show heaped up or flat centers. The agar m a third flask 



Jdges may be very thin and slightly (15) Rernove (/4) f-- th-t-^---^ 



serrated or may be raised well defined while hot add ^-^ /«• ° J ^ 



and smooth. al««holic solution of gentian violet 



Variants: 1.0 cc. of a 1:100,000 dilution to the infusion agar. 



of methyl violet in distilled water may be (16) Pour the conten s of the infus on 



added to each tube containing 10.0 cc. of agar gentian violet flask into the 



the medium and 5.0 cc. of the ascitic flask containmg the egg white, and 



g .^ then the egg yolk is added. 



Reference: Torrey and Buckell (1922 p. (17) Pour from one flask to another to 



j2g) insure thoro mixing. 



