70 ESSENTIALS OF BACTERIOLOGY 



PREPARATION OF NUTRIENT CULTURE-MEDIA 



(After the recommendations of the American Public Health Association) 



Materials. All water used should be distilled. 

 Fresh meat. 



Dried peptone, Witte brand. 



Best French gelatin, as free as possible from impurities. 

 Best commercial agar in threads. 

 Sugars, dextrose, lactose, and saccharose, all chemically 



pure. 



Glycerin, double distilled. 

 Azolitmin in place of litmus. 

 All other materials as nearly as possible chemically pure. 



Sterilization. Preferably in the autoclave and in small 

 containers, at 120 C., with 15 pounds pressure for fifteen 

 minutes. The sterilizer should be hot before the medium 

 is put in. 



Intermittent. For gelatin or sugar media a high tempera- 

 ture is not suitable. The media are placed in streaming 

 steam for thirty minutes on three successive days. 



Reaction. One-half per cent, solution phenolphthalein 

 (5 grams to i liter alcohol) is needed as an indicator. 

 The reaction should be +i per cent., i. e., i per cent, 

 alkaline solution required to make it neutral. 



Method of Obtaining Reaction. To 5 c.c. of medium add 

 45 c.c. water. Boil one minute. Add i c.c. solution 

 phenolphthalein. If the mixture is not tinted pink, the 

 medium is acid or neutral and requires the gradual addi- 

 tion of i : 20 normal sodium hydroxid solution until a 

 faint pink color remains. The soda should be added 

 while the mixture is hot or boiling. Calculate from the 

 amount of alkali used for the 5 c.c. how much will be 

 needed for the whole quantity of media and add the 

 same, using normal solution instead of i : 20 normal. 

 Example: If 2 c.c. NaOH will neutralize 5 c.c. media, 

 2 c.c. NaOH will neutralize 100 c.c., or 20 c.c. NaOH 

 will neutralize 1000 c.c. media, 



If the medium is very alkaline, hydrochloric acid must be 

 added to reduce to -j- i per cent. 



