242 PHYSIOLOGICAL CHEMISTRY 



first. The bacterial suspension now in the second beaker is again centrifugalized 

 in the same way and a third portion of bacteria-free sediment is separated. Fre- 

 quently a fourth serial centrifugalization is performed always if the third sediment 

 is of appreciable quantity. At all stages of the separation, small portions of the 

 dilute hydrochloric acid are used, so that the final suspension shall not be too vo- 

 luminous. Ordinarily it amounts to 125 to 200 c.c. At the same time, the final 

 amount of fluid should not be too small, as shown by Ehrenpfordt, 1 because the 

 viscosity accompanying increased concentration prevents proper and complete 

 sedimentation. 



To the final bacterial suspension an equal volume of alcohol is added and the 

 beaker set aside to concentrate. A water-bath at 50 to 6oC. is very satisfactory. 

 After two or three days, when the liquid is concentrated to about 50 c.c., the beaker 

 is removed and about 200 c.c. of alcohol are added. The beaker is covered and 

 allowed to stand at room temperature for 24 hours. At the end of this time the 

 bacterial substance is generally settled, so that most of the clear supernatant liquid, 

 of dark brown color, can be directly siphoned off without loss of solid matter. The 

 remainder is then transferred to centrifuge tubes, centrifugalized, and the remaining 

 clear liquid pipetted off. 2 The sediment consists of the bodies of the bacteria, and 

 is transferred to a Kjeldahl flask for nitrogen determination. This is the bacterial 

 nitrogen. Where a determination of bacterial dry substance is desired, the sedi- 

 ment of bacteria is extracted by absolute alcohol and ether in succession, trans- 

 ferred to a weighed porcelain crucible, and dried at io2C. to constant weight. 

 This dried sample is then used in the nitrogen determination. Our procedure 

 differs from that of MacNeal in that the bacterial dry matter is not determined. 

 A saving of about seven days' time and of considerable labor is accomplished by 

 this omission. 



Inasmuch as it has been shown by various investigators that such bacteria as 

 are present in the feces contain on the average about n per cent of nitrogen, the 

 values for bacterial nitrogen as determined by our method may conveniently serve 

 as a basis for the calculation of the actual output of bacterial substance. 



23. Quantitative Determination of Indol in Feces. Bergeim's Modification of the 

 Herter-Foster Method. 3 Principle. The feces is distilled from alkaline solution 

 to remove phenols. This distillate are again distilled from acid solution to remove 

 ammonia. The indol in the final distillate is treated with 0-naphthaquinone sodium 

 monosulphonate and alkali and the blue compound formed extracted with chloro- 

 form and determined colorimetrically. 



Procedure. Rub 30-50 grams of the fresh, well-mixed feces in a mortar with 

 water to a uniform consistency. Transfer to a wide mouth Kjeldahl flask of about 

 1000 c.c. capacity, rinsing mortar and neck of flask with distilled water to make 

 about 400 c.c. Add 5 c.c. of 10 per cent KOH solution and about 2 c.c. of paraffin 

 to decrease foaming. Distill with steam using ordinary Kjeldahl distillation ap- 

 paratus with good stream of water in the condenser. Heat carefully for a few 

 minutes until danger of foaming is past and then allow to boil vigorously. Distill 

 over 500 c.c. of liquid, bringing the volume of the fecal suspension down to about 

 100 c.c. toward the end of the distillation. 



1 Ehrenpfordt: Zeit. exp. Path. Ther., 7, 455, 1909. 



2 In later work (see Blatherwick and Hawk: Biochem. Bull., 3, 28, 1913) it was 

 found advantageous to centrifugalize with alcohol and ether in^succession before trans- 

 ferring the bacterial cells to Kjeldahl flasks. 



8 Herter and Foster: Jour. Biol. Chem., i, 257, 1906. Bergeim, Fishback, and Hawk: 

 Unpublished data. 



