METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA . 199 



Samples of Witte peptone occasionally do not contain tryptophan, 

 consequently each lot of peptone should be tested. When an especially 

 favorable sample is found it should be reserved for this purpose. Plain 

 neutral sugar-free broth is a better medium than Dunham's solution 

 for the indol test, and it should be employed for this purpose whenever 

 possible. 



Nitrate Broth. Add 10 grams of Witte peptone to one liter of water 

 and dissolve by boiling. Then add 0.2 gram chemically pure potas- 

 sium nitrate free from nitrites and filter. Sterilize in the autoclave. 

 The reaction does not require adjustment. 



Nutrient Gelatin Media. Ten grams Witte peptone and 5 grams 

 NaCl are added to 1 liter of sugar-free meat infusion 1 and dissolved by 

 boiling. When the ingredients are in solution, 100 2 grams of "Gold 

 Label" gelatin are added, a few leaves at a time, and stirred until 

 dissolved. The reaction is then adjusted to the desired degree and 

 verified after an additional five minutes' heating. The medium is 

 cooled to 50 C., and clarified with eggs, using two eggs for each liter. 

 Filter through a double layer of absorbent cotton in a large glass funnel 

 until clear, and sterilize. When sterilization is accomplished, cool 

 quickly and store in the ice-box. 



Nutrient Agar. (a) Dissolve 12 grams of powdered or shredded agar 

 in one liter of meat infusion by the aid of heat and add 5 grams NaCl 

 and 10 grams Witte peptone. Maintain a boiling temperature for at 

 least thirty minutes, or until the ingredients are completely dissolved; 

 restore the loss by evaporation, adjust the reaction, and filter through 

 a double layer of absorbent cotton in a large glass funnel. Pass through 

 filter until clear. It is frequently necessary to clarify agar with eggs. 

 After the reaction is adjusted, cool to 50 C. add two eggs beaten up 

 in water and mix thoroughly. Heat slowly to the boiling-point, boil 

 ten minutes, and filter through absorbent cotton; sterilize in the 

 autoclave. 



(6) Prepare "double-strength" meat infusion; 1000 grams of finely 

 comminuted lean meat are suspended in one liter of water; infuse in 

 the ice-box for twenty-four hours; heat to boiling and filter through 

 filter paper. Prepare nutrient meat infusion broth with this strong 

 infusion as a basis and adjust the reaction to twice the desired acidity 

 thus, if +1.0 is to be the final reaction, make the infusion broth 



1 Meat extract may be used in place of meat infusion, but the medium is not as satis- 

 factory for pathogenic bacteria. 



2 Use 120 grams gelatin during warm weather. 



