544 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



(e) Committee A.P.H.A. (1913). 



(1) Add 30.0 g. of agar to one liter of 

 cold water by sifting successive 

 small portions upon the surface 

 and allowing them to settle. 



(2) Add 10.0 g. Witte's peptone, 5.0 

 g. meat extract to (1) and boil 

 until solution is complete. 



(3) Neutralize to litmus by adding 

 NasCOs. 



(4) Distribute in 100.0 cc. quantities. 



(5) Prepare a 10.0% solution of 

 Na2S03 in water. 



(6) Prepare a 10.0% basic fuchsin 

 solution in 96% alcohol. 



(7) Add 2.0 cc. of (6) to 10.0 cc. of (5) 

 and steam for a few minutes in 

 the Arnold steamer. 



(8) Add 1.0 g. chemically pure lactose 

 to each 100.0 cc. of sterile (4). 



(9) Melt (8) and add 0.5 cc. of (7). 



(10) Pour in plates. 



(11) Sterilize (4) for 2 hours in stream- 

 ing steam. 



(f ) Robinson and Rettger. 



(1) Dissolve 25.0 g. powdered agar, 

 10.0 g. Peptone (American brand), 

 and 5.0 g. Liebig's meat extract in 

 1000.0 cc. water. 



(2) Make neutral to litmus. 



(3) Steam in the autoclave at 12 to 15 

 pounds extra pressure for 35 to 40 

 minutes. 



(4) Filter thru absorbent cotton and 

 cheese cloth. 



(5) Add NajCOs and heat for 10 

 minutes on boiling water bath. 

 Reaction 0.1 to phenolphthalein. 



(6) Add 10.0 g. lactose and 5.0 cc. of a 

 saturated alcoholic solution of 

 fuchsin to hot liquid. The me- 

 dium is now brilliant red. 



(7) Add 10.0 cc. of a 10.0% anhydrous 

 sodium bisulphite solution. 



(8) Distribute into large test tubes in 

 20.0 cc. lots. 



(9) Sterilize for 5 to 7 minutes at 10 

 pounds extra pressure. 



(g) Roddy. 



(1) Prepare a 3.0% extract agar. 



(2) Liquify (1) in the Arnold steam 

 sterilizer. 



(3) Dissolve 10.0 g. lactose in (2). 



(4) Adjust the reaction. Best results 

 are obtained when the medium is 

 just slightly alkaline to litmus. 



(5) Add exactly 3.0 cc. of a saturated 

 alcoholic fuchsin solution and 30.0 

 cc. of a 10.0% solution of anhy- 

 drous Na2S03 (or 3.0 cc. of a satu- 

 rated aqueous solution of Na2S03 

 may be added). 



(6) Tube. 



(7) Sterilize for 20 minutes on each of 

 3 successive days in an Arnold 

 sterilizer. 



(h) Committee A.P.H.A. (1917-1920). 



(1) Add 5.0 g. beef extract, 10.0 g. 

 peptone and 30.0 g. agar, dried at 

 105°C. for one hour before weigh- 

 ing to 1000.0 cc. of distilled water. 



(2) Boil on a water bath until all the 

 agar is dissolved and then make 

 up the water lost by evaporation. 



(3) Cool to 45°C. in a cold bath, then 

 warm to 65 °C. in the same bath 

 without stirring. 



(4) Make up lost weight. 



(5) Titrate and if the reaction is not 

 already between neutral and +1, 

 adjust to neutral. 



(6) Filter thru cloth and cotton until 

 clear. 



(7) Distribute in 100.0 cc. (or larger) 

 quantities in flasks. 



(8) Sterilize in the autoclave at 15 

 pounds pressure (120°C.) for 15 

 minutes after the pressure reaches 

 15 pounds. 



(9) Prepare a 10.0% solution of basic 

 fuchsin in 95.0% alcohol, allow to 

 stand 20 hours, decant and filter 

 the supernatant fluid. This is a 

 stock solution. 



(10) When ready for use prepare a 

 10.0% anhydrous sodium sulphite. 



(11) To each 10.0 cc. of (8) add 2.0 cc. 

 of (7) and steam for 5 minutes in 

 the Arnold or water bath. 



(12) To each 100.0 cc. of agar, add 1.0 

 g. of lactose, and dissolve in 

 streaming steam or on a water 

 bath and 0.5 cc. of (9). 



(13) Pour into Petri dishes and allow 

 to harden thoroly in the incubator 

 before use. 



