METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 197 



agate-ware boiler, add 5 grams of common salt (NaCl) and heat to 

 boiling. Dust 10 grams of Witte peptone over the surface and stir 

 until it is thoroughly dissolved. Restore the loss by evaporation and 

 adjust the reaction to the desired degree of acidity. Boil for five 

 minutes, verify the reaction and filter through filter paper until the 

 filtrate is perfectly clear. Sterilize in the autoclave. 



Meat Extract Broth. 1 To 1000 c.c. of meat extract (see page 190 for 

 preparation) in a tared agate-ware boiler, add 10 grams of Witte pep- 

 tone, dusting the peptone on the surface. Heat to boiling, restore 

 loss by evaporation and adjust the reaction. Continue the boiling for 

 five minutes, verify the reaction and cool to room temperature. 2 Filter 

 cold through filter paper until perfectly clear and sterilize. 



Nutrient Sugar-free Broth. Meat infusion contains small amounts 

 of muscle-sugar dextrose usually about 0.1 per cent. This sugar 

 is present in nutrient meat infusion broth prepared as outlined above. 

 It is frequently desirable to prepare meat infusion broth free from all 

 sugars. The dextrose is readily removed by fermentation with Bacillus 

 coli, adding a broth culture of this organism to the meat infusion before 

 it is heated and maintaining the infusion at 37 C., for eighteen to 

 twenty-four hours. The sugar which is attacked by Bacillus coli in 

 preference to the protein constituents of the medium 3 is quantitatively 

 removed. The organism must be killed as soon as the sugar is 

 exhausted, otherwise the protein constituents will be attacked. The 

 end of the fermentation may be judged with a fair degree of certainty 

 if one removes some of the infusion seeded with Bacillus coli to a fer- 

 mentation tube, kept at the same temperature, 37 C.; when gas is 

 no longer evolved the sugar is exhausted. Sugar-free broth contains 

 lactic acid, one of the products of fermentation of dextrose by Bacillus 

 coli. After the sugar is removed the medium is sterilized in the usual 

 manner, or made directly into sugar-free nutrient meat infusion broth 

 as outlined above. 



Nutrient Sugar Broth. One per cent, of dextrose, lactose, saccharose, 

 mannite, or other carbohydrate is added to nutrient sugar-free broth 

 immediately before filtering. Media containing sugars are best steri- 

 lized in the Arnold sterilizer on three successive days; the high tem- 

 perature of the autoclave tends to decompose carbohydrates. 



1 It is unnecessary to add salt to meat extract. 



2 A precipitate containing phosphates, soluble in the hot medium, settles out upon 

 cooling. It must be removed before the medium is used. 



3 See chapter on Bacterial Metabolism. 



