88 BULLETIN &6, U. S. DEPARTMENT OF AGRICULTURE. 
water at 40° C., thoroughly mix the material with a sterilized electric 
stirrer of the type commonly used at soda fountains for mixing eggs. 
One with a detachable plunger, which can be removed, dipped in 
alcohol and flamed, is best. Examine this mixed composite sample 
by the following methods: 
ENUMERATION OF VIABLE BACTERIA. 
Into a tared, sterile, glass-stoppered Erlenmeyer flask of 60 to 
100 cc. capacity, weigh approximately 5 grams of the mixed composite 
sample to the nearest hundredth of a gram, and dilute with 9 ce. of 
sterile physiological salt solution (0.85 per cent sodium chlorid) for | 
each gram of egg. Add about 2 grams of sterile glass beads or broken 
glass. Shake thoroughly until the mixture is homogeneous. Fur- 
ther dilutions are made in the regular way by transferrimg 1.0 ce. of 
the mixture to 9 cc. or 99 cc. dilution bottles of sterile physiological 
salt solution in the customary manner. From the appropriate dilu- 
tions prepare a series of plates with standard nutrient agar for 
counting after incubation at not less than 19° C. or more than 21°C. 
for 5 days. Perform all of these operations in duplicate. 
No count of bacteria is official unless based upon duplicate plates 
which agree closely and have between 30 and 300 colonies upon them 
when counted with a lens magnifying 34 diameters, or what opticians 
eall a three and one-half X lens. In case it is doubtful whether 
certain objects are colonies or dirt specks, examine them with a 
compound microscope. : 
ENUMERATION OF B. COLI. 
Inoculate lactose broth fermentation tubes, in duplicate, with ap- 
propriate dilutions of the sample, and make a quantitative determi- 
nation of the presence of members of the B. coli group. 
ENUMERATION OF TOTAL BACTERIA. 
In a tared clean flask of about 50 ec. capacity, weigh approxi- 
mately 5 grams of the well-mixed sample to the nearest hundredth 
of a gram, and dilute with twice the amount of physiological salt 
solution. Add sterile glass beads or broken glass, and mix thor- 
oughly till homogeneous. ‘Transfer a 0.01 cc. portion of this mixture 
to a microscopic slide by means of a capillary pipette of such bore 
that 0.01 cc. occupies from 1.2 to 2 centimeters, and the tip of which 
pipette has been ground to a truncated cone. Lay the slide on a 
piece of black paper upon which a square, 2 centimeters to a side, 
has been ruled with white ink. By means of a heavy platinum 
needle, bent into the shape of a hockey stick, spread the liquid 
evenly over the square within the white lines. A drop of distilled 
1 Standard Methods of Water Analysis of the American Public Health Association. 
