40 CULTIVATION OF BACTERIA. 



Beef-extract, 2 grams ; 



Peptone, 10 " 



Salt, 5 " 



Agar-agar, 10 " 



Water, 1000 c.c. 



The agar is chopped very fine and placed in hot water to 

 hasten its solution. The other ingredients are dissolved as 

 already described in the preparation of the bouillon and gela- 

 tin. The mixture is boiled until the agar is thoroughly dis- 

 solved. This may occur in half an hour or in two hours, 

 and during all this time the mixture is stirred steadily, as 

 agar burns very rapidly. After it is all in solution, neutral- 

 ize, cool, clarify with the whites of one or two eggs, and boil 

 again until the coagula are formed. 



Agar filters very slowly because it solidifies so rapidly. 

 To obviate this, the filter and the filtering fluid should be 

 kept as hot as possible. A hot-water funnel may be used for 

 this purpose ; or the filtering-stand is placed in the steam 

 sterilizer, over a low flame, which keeps the water hot without 

 causing it to boil. Only a little agar should be filtered at a 

 time, the remainder being kept hot. The first filtration may 

 be made through a finely woven cheese-cloth, after which 

 filter-paper should be used. The filtrate should be perfectly 

 clear and transparent. If all the steps in the process of pre- 

 paring the agar have been carried out carefully, the filtration 

 ought to be completed in about half an hour. Usually, how- 

 ever, it requires several hours, because students do not boil 

 the solution sufficiently in the first place, so that the agar is 

 not thoroughly dissolved. After filtration the medium is 

 transferred to sterilized test-tubes, about 10 c.c. in each tube, 

 and sterilized in the steam sterilizer for one hour on that day 

 and for half an hour on the next. Agar is solidified in the 

 inclined position, so as to have a large slanting surface for 

 inoculation. The water which usually collects on the surface 

 of solid media must not be removed. It prevents the medium 

 from drying too rapidly, and also favorably influences the 

 growth of the bacteria. 



Glycerin-agar-agar : Some species of bacteria will not grow 



