Plating and Counting 133 
second dilution and place in the third bottle. One c. ec. 
of this transferred to the plate will give a dilution of 
1 : 1,000,000. 
Checks.—With a sterile pipette, place 1 c. c. of 
the dilution water in a sterile plate, and add the usual 
amount of media. Also pour 10 c. c. of media 
into a sterile, empty petri dish. If litmus is used, 
make a third check with 2 ¢. c. of litmus and 10¢. ¢. 
of the media. ._In this way the state of the material 
may be determined. The following scheme from the 
Iowa Bacteriological Laboratory Report will be of 
value in estimating dilutions: 
After the diluted milk is in the petri dish, if the litmus is 
wanted, add 2 c. c. with a sterile pipette. If not desired, this 
may be omitted. Melt the tubes of agar or gelatin, cool and 
maintain at a temperature of 40° C. Finally, add 10 c. c. of the 
nutrient media, either agar or gelatin as desired, being careful to 
pass the mouth of the tube through a flame before pouring it. 
Give the dish a revolving motion, to mix the diluted milk and 
media, and then allow it to harden. Agar should be incubated’ 
at 37° C. for forty-eight hours, while gelatin needs a temperature 
of 20° C. for five days. When ready to count, place the petri dish 
over a counting-plate and, with a hand lens, count the number of 
colonies. If a counting-plate is not available, lines may be made 
across the bottom of the plate with a blue pencil, for marking 
glass to aid in counting. Each colony represents an original 
organism. Multiply the number of colonies by the dilution, and 
the result is the approximate number of organisms in the sample. 
Repeat this with six plates, and take the average of them as the 
final count. 
