146 BACTERIOLOGY. 
and heat until the egg albumen has become coagulated. Filter while 
hot through a filter paper previously moistened with hot water, and 
preserve in a sterilized flask or sterilized test tubes which are stopped 
with plugs of cotton wool. When cold the product must be perfectly 
clear and transparent. 
How is nutrient agar agar prepared ? 
The beef bouillon is.prepared in the usual way. To 1000 grams of 
this, which has been neutralized, add 10 to 15 grams in small pieces of 
agar agar (a vegetable gelatin). Place the whole in a Florence flask 
or a porcelain-lined vessel, marking the level at which the mixture 
stands, and add about 250 cc. of water. Boil the mixture slowly with an 
occasional stirring for two or three hours. If the level of the fluid falls 
below the original mark add more water. After boiling the mixture 
for a sufficient time, put the vessel containing it into a dish of cold 
water and stir continually. When the temperature has fallen to 68° 
or 70° C., add the whites of two eggs dissolved in 50 ce. of water and 
boil slowly for half and hour. Then filter quickly through heavy filter 
paper previously wet. After filtering, the mixture is poured into 
sterilized test tubes afterwards plugged with cotton wool. These tubes 
are placed in an oblique position so that as the mixture solidifies more 
surface is exposed for purposes of inoculation. Although when hot 
the filtered product is perfectly clear, on cooling it usually becomes 
rather opaque. 
How are culture tubes prepared with blood serum? 
Blood may be procured from small or large animals and received in 
thoroughly sterilized jars. After standing in a cool place for 24 hours 
the blood will have formed a clot and the serum will be separated from 
it. This is removed from the jars and allowed to stand in order that 
all corpuscular elements of the blood present may settle. The clear 
serum is drawn off with a pipette and put into sterilized test tubes plug-_ 
ged with cotton wool. The serum is sterilized at a low temperature by 
the discontinuous method extending over a period of four or five days. 
The tubes with their contents are then laid in a desiccator ina slant- 
ing position and kept at a constant temperature of from 75° to 78° C. 
until the serum has solidified. The tops of the cotton plugs are 
burned off to destroy any mould that may be in them, and covered with 
rubber tissue to prevent further evaporation. If human blood serum 
is wanted, the blood may be obtained from a human placenta that has 
been carefully washed, or from hydrocele or ascitic fluid. 
