8o 



METHODS OF CULTIVATION OF BACTERIA. 



the cotton plug by a sterile rubber stopper before opening up 

 the closed arm. 



The composition of the medium is, of course, of great impor- 

 tance, and in testing the effect of a bacterium on a given sugar 

 it is essential that this sugar alone be present. 

 The first method is usually used with ordinary 

 gelatin, and the gas-formation in most cases 

 results from fermentation of the glucose 

 naturally present in the medium from trans- 

 formation of the glycogen of muscle. (It is 

 only a more deHcate method of demonstrating 

 what sometimes occurs along the line of 

 growth in an ordinary gelatin stab-culture.) 

 The amount of glucose naturally present, 

 however, varies much, and therefore glucose 

 should be added to the medium if the effects 

 on this sugar are to be observed. When 

 other sugars — lactose, mannite, etc. — are to 

 be tested, these should be added either to a 

 simple peptone solution as Durham recom- 

 mends, or to bouillon previously freed from 

 dextrose as described below — fermentation being observed by 

 either of the methods (2) or (3). 



To obtain a " dextrose-free " bouillon, Smith advises that the beef infusion, 

 prepared by extracting in the cold or at 60° C. for twelve hours, be inoculated 

 in the evening with a rich fluid culture of B. coli and placed in the thermostat 

 over night. Early the following morning the infUsion, covered with a layer of 

 froth, is boiled, filtered, peptone and salt added, and neutralisation and sterili- 

 sation carried out as usual. As a test for the complete removal of the sugar, 

 a fermentation tube of the broth when inoculated with B. coli will no longer 

 give a growth in the closed arm of the tube, the fluid there remaining perfectly 

 clear. When the various sugars are added to such a broth, it is strongly 

 advised to sterilise by the intermittent method, for the heat of the autoclave 

 is almost sure to produce chemical changes in the sugars through the presence 

 of alkali and other constituents in the medium. 



The Observation of Indol formation by Bacteria. — The for- 

 mation of indol from albumin by a bacterium sometimes con- 

 stitutes an important specific characteristic. To observe indol 

 production the bacterium is grown preferably at incubation tem- 

 perature on a fluid medium containing peptone. The latter may 

 either be ordinary dextrose-free bouillon or peptone solution 



Fig. 45. — Hill's modi- 

 fication of Smith's fer- 

 mentation tube. 



