248 INTESTINAL BACTERIA 
a portion of the 1: 100 suspension of feces is diluted ten times, and a 
portion of this is drawn up to the mark 1 in the capillary of a dilution 
pipette, ordinarily used in estimating the white blood cells. This is 
diluted to the mark 11 with a dilute solution of methylene blue in physio- 
logical-salt solution. (The staining solution consists of methylene 
blue, 1 gm.; glycerine, 25 c.c.; distilled water, 75 c.c. A few drops 
of this are mixed with 10 c.c. of 0.8 per cent salt solution until the mix- 
ture is well colored, but not too opaque. This mixture is used as the 
diluting fluid. A little practice will show the proper depth of color to 
be employed.) The suspension is thoroughly mixed in the bulb by 
shaking and rolling in the usual manner. Several drops are blown out 
and then a very small drop is placed in the center of the circular elevated 
portion of the slide, which has been previously thoroughly cleansed by 
washing in distilled water and alcohol. A clean, thin, ground cover-slip 
is made slightly moist by breathing upon it and is immediately placed 
upon the slide. Slight pressure upon it causes the Newton color rings to 
appear and these remain after the pressure is removed if the preparation 
has been properly made. The slide is allowed to stand one to two hours 
to allow the bacteria to settle. Then the bacteria in fifty small squares 
upon the marked scale are counted microscopically, the No. 7 Leitz 
objective and No. 3 ocular being used. The calculation is simple as an 
example will show. 
“ Example. B252, Subject H, Julv 15, 1908. Fifty squares contain 
400 bacteria. Therefore the average per square is 8.0 bacteria. One 
small square=1/4000 cmn. One c.mn. contains 8x4000=32,000 
bacteria. One cubic centimeter contains 32,000 1000 = 32,000,000 
bacteria. Pipette dilution is 1:10. 32,000,000 10=320,000,000 bac- 
teria per cubic centimeter of 1/1000 suspension. One cubic centimeter 
of 1/1000 suspension is equivalent to 1 mg. feces. Therefore, there are 
320,000,000 bacteria per milligram feces. 
‘“ Counting by this method is an exacting process and much practice 
is required to see all the bacteria present. Careful adjustment of 
the light is important and best results have been obtained by illumina- 
tion with Welsbach light; constant focusing through the different layers 
is necessary. Perhaps the greatest source of error is the difficulty of 
distinguishing accurately the bacteria. Skill in this is acquired only by 
practice. The method has the advantage of simplicity. The results 
of the authors seem to conform to those of Winterberg in that high 
dilutions give a relatively high count.” 
MacNeal’s (et al.) Modification of the Eberle-Klein Method for 
Determining the Number of Bacteria in Feces. Eberle, working in 
