3 12 LABORATORY WORK. 



more for twenty-four hours and then steam a third time. If properly 

 sterilized these media will keep indefinitely. 



No. 2. Determination of the Number of Bacteria in Water. Melt three 

 tubes of agar as above made in hot water and cool to about 1250 (5oC.) 

 With a sterilized pipette place a cubic centimeter of water from any source 

 in each of three Petri dishes (Fig. 61.), and pour the agar from one of the 

 melted tubes into the Petri dish. Replace the cover again at once and by 

 gentle agitation, thoroughly mix the agar in the dish with the water first 

 added. Do not do this violently enough to spill or throw the agar up on 

 the sides of the plate. After mixing set on a level table to harden and 

 then place in the culture oven. These preparations are called agar plates. 

 After twenty-four hours the plates will be seen to be dotted over with 

 colonies, each supposed to come from a single bacterium. Count the 

 number of colonies; this will give the number of bacteria in the original 

 c.c. of water that have been able to grow in the medium. 



FIG. 61. A petri dish for making "plates." 



No. 3. Water Blanks. Place in a considerable number of test-tubes 

 9 c.c. of water. In a series of small flasks or bottles place 99 c.c. of water 

 (i.e., add 100 c.c. and then remove i c.c.) After plugging tightly with 

 cotton, place in the autoclave and sterilize at 15 pounds for one hour. It is 

 very desirable to have test-tubes and bottles used for these water blanks 

 with a mark etched upon them at the 9 c.c. and 99 c.c. level. When they 

 are to be subsequently used for dilution care should be taken to see that 

 they are filled, exactly to the mark, since evaporation frequently with- 

 draws some of the water, and unless they contain the exact quantity, errors 

 will be introduced. 



No. 4. Determination of the Bacteria in Milk. Milk commonly contains 

 so many bacteria that it must be diluted before the bacteria are determined. 

 The amount of dilution needed will vary widely with the age of the milk. 

 For most market milk a dilution of 1,000 will serve. Proceed as follows: 

 Into a 99 c.c. water blank place i c.c. of the milk to be tested. Shake 

 vigorously so as to distribute the bacteria uniformly. With a second pipette 

 transfer i c.c. of this mixture to a 9 c.c. water blank and again thoroughly 

 mix. With a third pipette place i c.c. of this in a Petri dish and then pour 

 upon it the contents of an agar tube. Agitate as in the last experiment, 

 allow to harden and incubate in the oven for twenty-four hours. Use a 

 counting plate in counting the colonies. If they are too numerous to 



