BACTERIA IN FECES 253 
and transferred to a beaker. The sediment remaining in the tube is 
again rubbed up with a glass rod with the addition of further amounts 
of dilute acid, and again centrifugalized for one-half to one minute. The 
supernatant liquid is pipetted off and added to the first, the same 
pipette being used for the one determination throughout. A third por- 
tion of the dilute acid is then added to the sediment, which is again 
mixed by stirring and again centrifugalized. All the washings are added 
to the first one, and, during the process, care is taken to wash the 
material from the wall and mouth of the centrifuge down into it. Finally, 
when the sediment is sufficiently free from bacteria, the various remain- 
ing particles are visibly clean, and the supernatant liquid, after cen- 
trifugalization, remains almost clear. This is removed to the beaker 
in which are now practically all of the bacteria present in the original 
portion of feces, together with some solid matter not yet separated. In 
the centrifuge tubes there is a considerable amount of bacteria-free solid 
matter. 
“The suspension is now transferred to the same centrifuge tube, 
centrifugalized for a minute, and the supernatant liquid transferred to 
a clean beaker by means of the same pipette. The tube is then refilled 
from the first beaker and thus all the suspension centrifugalized a second 
time. The beaker is finally carefully washed with the aid of « 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 portions 
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. Frequently 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 voluminous. 
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, 
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 
60° 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 twenty-four hours. At the end of this time 
