88 BULLETIN 846, 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 cc. 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 transferring 1.0 cc. 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 Sh diameters, or what opticians 

 call 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 OP 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. 1 



ENUMERATION OP TOTAL BACTERIA. 



In a tared clean flask of about 50 cc. 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. 



