CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



543 



(2) Add 10.0 g. Witte's peptone and 

 3.0 g. Liebig's meat extract to 

 (1) and cook in a double boiler for 

 one hour. 



(3) Make faintly alkaline to litmus by 

 cautious addition of NaOH. 



(4) Cook 15 minutes to set the 

 reaction. 



(5) Filter thru absorbent cotton. 



(6) Distribute in 100.0 cc. lots in 

 flasks and sterilize in the auto- 

 clave. 



(7) Prepare a 10.0% solution of 

 fuchsin in 96. 0% alcohol. 



(8) Prepare a 10.0% solution of sodium 

 sulphite in water. 



(9) Add 1.0 cc. of (7) to 10.0 cc. of (8) 

 and heat in the Arnold sterilizer 

 for 20 minutes. 



(10) Add 1.0%, lactose (which must be 

 chemically pure) to (6). 



(11) Heat in Arnold sterilizer until the 

 agar is melted and the lactose 

 thoroly distributed in it. 



(12) Add (9) (decolorized fuchsin solu- 

 tion) in the proportion of 1.0 cc. 

 to each 100.0 cc. of the medium. 



(13) Mix thoroly and pour into sterile 

 plates. 



(14) Allow to harden (with covers 

 removed) in the incubator for 30 

 minutes. The plates are now 

 ready for inoculation. 



(d) Kinyoun and Deiter. 



(1) Mix 80.0 g. Witte's peptone, 40.0 

 g. NaCl and 160.0 g. powdered 

 agar with sufficient water to make 

 a smooth paste. 



(2) Add suflBcient water to make 8000.0 

 cc. 



(3) Add 80.0 g. Liebig's meat extract 

 (not necessary to mix). 



(4) Place in the Arnold sterilizer and 

 steam until solution is complete. 

 Usually requires about an hour. 



(5) Cool to 55°C. and titrate to +1 

 using phenolphthalein as an 

 indicator. 



(6) Place in tall beakers, steam an 

 hour and allow to solidify. (Allow 

 the beakers to stay in the steamer 

 over night.) 



(7) Remove the solid agar from the 

 beaker and cut away all the agar 

 containing sediment. 



(8) Cut the clear agar in small pieces, 

 replace in the beakers and melt 

 again. 



(9) Cool to 55°C. 



(10) Place 18 lots of 10.0 cc. each in an 

 equal number of sterile test tubes 

 and divide the tubes in two series. 



(11) To one series add 0.01, 0.03, 0.05, 

 0.07, 0.09 and 0.1 cc. of normal 

 HCl. 



(12) To the other series add 0.0, 0.01, 

 0.03, 0.05, 0.07, 0.09, 0.1, 0.3, 0.5, 

 0.7 and 0.9 cc. of 2.5% solution of 

 NasCOs. 



(13) To each tube of (U) and (12) add 

 1.0 cc. of a 10.0% solution of 

 crystalline lactose, 1.0 cc. of a 

 2.5% solution of NazSOs, freshly 

 prepared, and 1.0 cc. of a half 

 saturated alcoholic solution of 

 basic fuchsin. 



(14) Pour each tube into sterile petri 

 dish and allow to harden. 



(15) Inoculate one-half the surface of 

 each plate with a 24 hour broth 

 culture of typhoid bacilli, and the 

 other half of each plate with a 

 similar coli culture. 



(16) Incubate for 24 hours. 



(17) The plate showing most typical 

 luxuriant growth of both the coli 

 and typhoid is chosen as the 

 proper amount of acid or alkali 

 to add to the remainder of the 

 medium. 



(18) Add the necessary amount of acid 

 or alkali to the entire lot of agar 

 (9). 



(19) Distribute in 100.0 cc. lots m 



(20) Sterilize in the Arnold for an hour 

 on two successive days. 



(21) Store until ready for use. 



(22) When ready for use add 1.0 g. 

 crystalline lactose to each flask of 

 melted agar. 



(23) When the lactose has dissolved 

 add 5.0 cc. of a 5.0% solution of 

 anhydrous NaaSOs, freshly pre- 

 pared and still hot to each flask. 



(24) Add 1.0 cc. of a half saturated 

 alcoholic solution of basic fuchsin 

 to each flask. 



(25) Mix well. 



(26) Pour into plates. 



