336 LABORATORY WORK 
position and set aside for future use. Keep this and all other culture media in a 
cool dark place until used. 
Bouillon.—To make bouillon the procedure is as above, except that agar is not 
added. The other half of the dissolved mixture of water, peptone, and beef extract, 
in (a), has its acidity adjusted, if necessary, in exactly the same way as above 
described, and, after a boiling (the egg white is not necessary here) the bouillon is 
filtered through filter-paper, placed in tubes, and sterilized in exactly the same 
manner as agar medium. 
If it is not convenient to use the autoclave, either of these may be sterilized by 
discontinuous heating as follows: Place the tubes and flask in a steam sterilizer and 
steam for one-half hour (Fig. 59). Set aside for twenty-four hours and then steam 
for another half-hour. Set aside once 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 125° (s0°C.). With a 
Fic. 60.—A petri dish for making ‘‘plates.” 
sterilized pipette place a cubic centimeter of water from any source in each of 
three Petri dishes (Fig. 60), 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. 
No. 3. Water Blanks.—Place in a considerable number of test-tubes 9 c.c. of 
water. Ina series of small flasks or bottles place 99 c.c. of water (i.e., add roo c.c. 
and then remove ic.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 
withdraws some of the water, and unless they contain the exact quantity, errors will 
be introduced. 
