ZOOLOGY AND BOTANY, MICROSCOPY, ETC. 
527 
100 grams of beef. On the following day the liquid is separated from 
the meat by straining it through a coarse linen. The simple beef- 
infusion thus obtained should be equal in quantity to the amount of 
water added ; if it is not the deficiency can be restored by the addition 
of distilled water. To the beef-infusion is added 1 per cent, peptone, 
1/2 per cent, sodium chloride; and if it is desirable to make it 
alkaline, a sufficient quantity of a normal solution of sodium carbonate 
to give it a weak alkaline reaction. The liquid is then boiled for thirty 
minutes in a water-bath, cooled, filtered, and distributed in Erlenmeyer 
flasks plugged with cotton-wool. If only a small quantity of agar is to 
be made at once, 250 ccm. is found to be a very convenient quantity to 
put in each flask. It is then sterilized by boiling for one hour each day 
for three consecutive days. It need not be sterilized if it is desirable to 
prepare the agar at once. As the beef- infusion-peptone is also employed 
as a liquid medium in the cultivation of bacteria, very little time is lost 
in preparing an extra quantity of this liquid to be used in making the 
agar. 
(2) The preparation of the agar . — To an Erlenmeyer flask (a glass 
beaker or agate or iron vessel may be used) containing beef-infusion- 
peptone, as prepared above, 1 per cent, of very finely chopped agar is 
added. The flask is then placed in a water-bath and boiled vigorously 
for two hours. At the end of that time the agar is dissolved, and the 
liquid is allowed to cool. When a temperature of 40-45° C. is reached, 
the white of egg is added in the proportion of one egg to 250 ccm. of the 
liquid. After the albumen is thoroughly mixed with the liquid agar it 
is returned to the water-bath and again boiled for two hours. It is of 
much importance that the albumen is evenly distributed throughout the 
mass before it is coagulated. It is now ready to be filtered. The egg 
albumen is coagulated in very firm masses, leaving the liquid perfectly 
clear. The coagulum is removed by filtering the liquid through fine 
Japanese filter-paper or a layer of absorbent cotton, as a 1 per cent, 
solution of the agar does not pass readily through ordinary filter-paper. 
Should a weaker solution of the agar (1/2 to 3/4 per cent.) be desired, 
its filtration can be accomplished by the ordinary method. A hot 
filtering apparatus is not necessary. The clear filtration is now ready 
for distribution in sterile cotton plugged tubes. 
The agar is sterilized by discontinuous boiling in a closed water- 
bath for three consecutive days. If small tubes have been used con- 
taining not more than 7 ccm. each, five minutes’ boiling each day is 
sufficient. If larger tubes are used, they should he boiled for a longer 
time. Or it may be sterilized by steaming each day for from five to ten 
minutes after the agar has become liquefied for the same number of days. 
After its sterility has been tested by allowing it to stand in an incubator 
for several days, it is ready to be stored until required for use. It has 
been customary in this laboratory, in order to prevent the evaporation 
of the agar by long standing, to dip the lower end of the cotton-plugs in 
hot sterilized paraffin, and to store the tubes in a cool, moist chamber.” 
