METHODS FOR MICROSCOPIC STUDY OF BACTERIA 22b 
commercial trypsin powder. When the biuret test is positi^•e/ the 
mixture is made slightly acid with acetic acid and brought to the 
boiling-point, and simmered for ten minutes. Add 5 gm. XaCl- for 
each 1000 cc. and filter through filter paper. Adjust the reaction to 
pH 7.4. 
The broth thus prepared may be sterilized, or converted into nutrient 
agar by dissolving in it 15 gm. agar for each 1000 cc. 
Nvtrient Sugar-free Broth. —Me&t infusion contains small amounts 
of muscle-sugar— glucose— 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 glucose 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,^ 
is quantitatively removed. The organism must be killed as soon as 
the sugar is exliausted, 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 fermentation 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 
glucose by Bacillus coli and some acetic acid as well. 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 aboAC. 
Many bacteria may be conveniently differentiated through their 
respective abilities to utilize certain carbohydrates for energy. In bac- 
teriological terminology, bacteria "ferment" certain carbohydrates. 
Sugars, starches or alcohols should always be dissolved, or suspended 
in distilled water, and sterilized in this form. Proper amounts of such 
concentrated, sterile solutions are added to sterile sugar-free broth, 
with proper precautions to bring about a final concentration of 1 per 
cent. The reaction of such a medium should be neutral or slightly 
acid, never alkaline. In faintly alkaline solutions, the hexoses, as 
glucose, undergo a gradual transformation into a considerable variety 
of products, including acids, and into an equilibrium mixture of glucose, 
fructose and mannose.'* The optical rotation also falls practically to 
zero under such conditions. Many observations upon the "fermenta- 
bility" of sugars by bacteria therefore will have to be restudied under 
carefully controlled conditions before they are acceptable. 
' Biuret test: 5 cc. medium and 0.1 cc. of 5 per cent copper sulphate solution are mixed 
thoroughly and then 5 cc. of normal NaOH solution is added. When the pink color 
gives way to a true blue color, the digestion is stopped. 
2 Gordon, Hine and Flack: British Med. Jour., 1916, ii, 678; add also 0.125 gm. CaCh 
to the solution. 
' See chapter on Bacterial Metabolism. 
* Lobry de Bruyn and von Eckenstein: Ber. d. deutsch. chem. Gcsell., 1S95, 28, 
.3078. Fernbach and Schoen: Ann. Inst. Pasteur, 1914, 28, 692, 
15 
