FECES 245 



with the aid of a rubber-tipped glass rod, the second sediment in the centrifuge 

 tube is washed free of bacteria by means of this wash water and by successive por- 

 tions of the dilute acid, and the supernatant liquid after centrifugalization is added 

 to the contents of the second beaker. The second clean sediment is added to the 

 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 trie 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 are distilled from alkaline solu- 

 tion to remove phenols. This distillate is again distilled from acid solution to 

 remove ammonia. The indol in the final distillate is treated with /5-naphthaqui- 

 none sodium monosulphonate and alkali and the blue compound formed extracted 

 with chloroform 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- 



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



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

 advantageous to centrifugalize with alcohol and ether in succession before transferring 

 the bacterial cells to Kjeldahl flasks. 



3 Herter and Foster: Jour. Biol. Chem., i, 257, 1906. Bergeim: Jour. Biol. Chem. 

 32, 17, 1917. 



