12 General Bacteriology. 
b. 1)-4) Same as a-d in the preparation of bouillon (IV.), except that only one- 
half (500 ec.) of the amount of water is added to the beef or extract. 
5) Add 1% peptone and 0.5% salt. 
6) Heat until peptone is dissolved. 
7) Neutralize. 
8) Mix a. and b. (in case beef is used it will be necessary to cool a. to about 60°. 
C. before mixing). 
9) Boil 5 minutes and restore weight. 
10) Test reaction. : 
11) Addition of egg will be necessary only where extract is used. 
12) Filter as in case of gelatin, (IV. m.) 
13) Tube. 
14) Sterilize in steam for 15 minutes on three suc- 
cessive days or in autoclave for 20 minutes at 120° C. 
After the last sterilization place most of the tubes in a 
sloping position to harden (fig. 4), these are known as 
agar slopes. Those solidified horizontally can be used for plate cultures. 
15) Label. 
REFERENCES. A. 100; H. 43; M. & R. 48; McF. 129; N. 235; P. B.C. 27; S. 
43; Journal of Applied urossony: 1898, 1; 106. 
SpeciaL Directions. Use meat extract, make 1 liter, fill 25 tubes and after last 
sterilization incline 20 of them. Place the remainder in flasks and sterilize. 
Fic. 4. Method of sloping agar. 
EXERCISE IX. PREPARATION OF POTATOES. (BOLTON.) 
GENERAL DIRECTIONS. 
a. Select a number of rather large test-tubes (15020 mm.) place a 
small wad of absorbent cotton in the bottom of each (fig. 5 a), plug and We) 
sterilize as usual. 
b. Wash a large potato, then with a cork borer slightly smaller than 
the test-tubes punch out cylinders about 5-6 cm. long. 
c. Divide these diagonally and trim to shape indicated in fig. 5 b. 
d. Add a few drops of distilled water to each test-tube and place 
pieces of potato in position. 
e. Sterilize on three consecutive days for 30 to 45 minutes. 
Unless the tubes are to be used immediately, they should be sealed. 
(XI.) The dark color can be prevented by immersing the pieces between ¢ Fic.5. Bolton’s 
and d@ in running water for from 12-18 hours. Potatostubes 
REFERENCES. A. 104; M. & R. 54; McF. 134; N. 183; P. 216; P. B.C. 28; S. 47. 
SpeciaL Directions. Prepare 15 test-tubes of potato, sterilize, label, and seal with 
paraffin. (XI. 2.) 
EXERCISE X. PREPARATION OF WATER-BLANKS. 
GENERAL DtREcTIONS. Water-blanks are prepared by placing exactly 10 ce. of a 
physiological salt solution (6 poe: per 1,000 ce. of water) in test-tubes and sterilizing in 
autoclave 15 minutes at 120 ° C., or in steamer 15 minutes on three successive days. 
SPECIAL DIRECTIONS. Prepare and sterilize 10 water-blanks. 
