LABORATORY AND TEACHING METHODS Oil 



Cellulose solution, c.c 500 



Agar, grams 10 



Nutrient solution, as follows: 



Potassium phosphate (dibasic), gram i 



Magnesium sulphate, gram i 



Sodium chloride, gram i 



Ammonium sulphate, grams 2 



Calcium carbonate, grams 2 



Tap water, c.c 1000 



Chestnut Twig Agar. — To 275 grams of one- or two-year-old chestnut branches 

 add 500 c.c. of distilled water and boil over an open flame for one-half hour. Filter 

 the juice and make up to 550 c.c. with distilled water. To 50 parts of this infusion 

 add 100 parts of distilled water and 2 per cent, of agar flour. Steam for one-half 

 hour, filter, tube and autoclave for fifteen minutes at ii5°C. 



LESSON 17 



Culture Medium. — Winogradsky employed for culturing upon solid media a 

 mineral gelatin. A solution of from 3 to 4 per cent, of silicic acid in distilled water 

 is placed in flasks. By addition of the following salts to such a solution gelatiniza- 

 tion occurs. 



(a) Ammonium sulphate, gram 04 



Magnesium sulphate, gram 0.05 



Calcic chloride a trace 



{b) Potassium phosphate, gram o. i 



Sodium carbonate, gram 0.6,0.9 



Distilled water, c.c 100 . o 



The sulphates and chloride are mixed in 50 c.c. of distilled water, and the latter 

 substance in the remaining 50 c.c. in separate flasks. After sterilizing and cooling 

 these are all mixed and added in small quantities to the silicic acid. Upon this 

 medium, it is possible to subculture a pure growth from the film at the bottom of 

 the flasks in which the nitrous organism is first isolated (c/. Newman, George: 

 Bacteria, pp. 154-157). 



Isolation of the Nitric Organisms. — Nitrobacter develops freely in solutions to 

 which no organic matter has been added; indeed, much organic matter will prevent 

 its growth. Winogradsky used the following medium to isolate it: 



Water, c.c 1000 o 



Potassium phosphate, gram i . o 



Magnesium sulphate, gram 0.5 



Calcium chloride a trace 



Sodium chloride, grams 2.0 



About 20 c.c. of this solution is placed in a flat-bottom flask and a little freshly 

 washed magnesium carbonate is added. The flask is closed with cotton-wool, and 



