CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 527 



(7) Titrate with N/1 NaOH to neutral (2) Mince finely. 



point with phenolphthalein. (3) Allow to macerate in water over- 



(8) Check the titre by repetition using ^l^^^' . ^ , ,. .^, „ 

 N/20 NaOH-5 cc. should require (4) Heat in a water bath with con- 

 1 to^2 cc. to display color while stant stirring until the proteins 

 , have been coagulated. 



, ° ^ (5) Filter thru coarse cloth. 



Sterilization: Sterilize in autoclave at (6) Make up to volume, 750.0 cc, with 



10 pounds for 30 minutes. hot distilled water^ 



Use: Cultivation of gonococci. The (7) Add to the fluid, while hot, 



author reported that media with 1.0%, 20.0 g. peptone and 3i) g. di- 



2 0%, 3.0% or 4,0% agar were also tried hydrogen sodium phosphate, 



with and without glucose and varying (8) Allow the temperature to fall to 



amounts of NaOH. The concentrations 40°C. , j. , ,, 



given were found to be the most satis- (9) Estimate and adjust the reaction 



^ to pH 7.6. 



(a) Clark prepared the medium as (10) Add 30.0 g. agar Previously dis- 



follows- solved in 250.0 cc. distilled water 



(1) Remove and discard tunica vagi- and finally 5.0 g. glucose, 

 nalis from fresh beef testicles and (H) Mix. 



rinse in running water and grind. (12) Distribute in test tubes. 



(2) Mix ground testicles (500.0 g.) (13) Sterilize at 120°C. _ 



with equal weight of distilled (14) Rotate the tubes to mix before 



water (500.0 cc.) at room tempera- allowing to solidify 



ture, and infuse over night. References : Hall (1916 p. 351), Clark (1920 



(3) In the morning heat to 50°C. for p. 100), Harvey (1921-22 p. 98). 

 one hour. Heat in steam kettle or 



double boiler, stirring, until the 1726. Kligler's Lead Acetate Infusion Agar 



proteins are coagulated and tend constituents: 



to collect in large flocculi. Do not ^^ ^^^^^ infusion agar (agar 



heat over free flame. ^^ ^^ jq q ^ -^ 1000.0 cc. 



(4) Strain thru coarse cloth and, if ^ Glucose 0-1% 



necessary, add distilled water to ^- ^^^^ acetate! .'.'.'.'." 0.05% 



750 cc 



(5) Dissolv; 20^0 g. Peptone (Parke ^^^^''^f^^^^^ ^^^, i,,^,,i,, ,g,, i, the 

 Davis) and 3.0 g. NaH.PO. + H.O ^J^^ ^^^_ ^^^ ^ ^^ ^^ ^oor, ^^^^^ 



m warm (4, (40 C. however. Details of method not 



(6) Adjust while warm to pH = 7.4 to 



7.8. Heat to boiling and adjust The^'Veaction should be between 



more precisely. dH = 7 2 to 7 6 



(7) Soak 25 to 30.0 g. agar in 250.0 cc. ^^.^^^^^^ ^^^ ^ ^^^^^^ ^^^ 05^^ 

 distilled water, and autoclave long ^^^^ ^^^^^^^ .^ ^^^^^ separately. 



.c^ T.T^ u f m T TfiT" (4) Cool sterile melted agar to 60° and 



(S) Add melted (7) to (6). ^^^ ^^^ ^^^^.j^ ^^^^^-^^ ^f gl^.^se 



(9) Add 5.0 g- glucose- ^ .^ . ^ and lead acetate to the agar under 



(10) Mix thoroly and distribute mto conditions, 

 desired containers ^^^^^^ 



(11) Autoclave at lo pounds for [gj ^^.^^ate to test sterility. 



(12) RoTaTe tubes to mix before Sterilization: Sterilize (2) and (3) sepa- 

 slanting. rately-method not given 



(b) Harvev gave the following method of Use: Differentiation of typhoid and para- 

 preparation: typhoid group. Author reported that 

 (1) Remove the tunica vaginalis of the stab cultures showed the following 

 bulls testicles. reactions: 



