ISOLATION OF SPECIES. 8% 
(c) Add ¥%4 c.c. of chloroform, or 5 per cent. car- 
bolic acid, to each tube, shake thoroughly, and 
filter. 
(d) Add the filtrate, now containing no living 
bacteria, to tubes of sterile gelatin. Note the 
liquefaction that takes place, caused by the fer- 
ment produced by the bacteria in the first set of 
tubes, 
IX. ISOLATION OF SPECIES. 
Given a bouillon culture, containing three species 
of bacteria, to isolate the species.' 
(a) Liquefy three gelatin or agar tubes and 
number 1, 2, and 3 respectively.’ 
(2) Transfer a minute loopful of the bouillon cult- 
ure to tube No. I. 
(c) Shake thoroughly, and transfer 2 loopfuls 
from tube No. 1 to tube No. 2. 
(a) Shake and transfer 3 loopfuls from tube No. 
2 to tube No. 3. 
(e) Flame the lips of the tubes and pour their 
contents into sterile Petri dishes. — 
(/) Examine in twelve to twenty-four hours. 
(g) Select the dish in which the colonies are 
well developed, and in which they have not run 
together ; look for three kinds of colonies. 
If more than three kinds are present, it shows 
1 This method is applicable to the separation of species 
from any fluid. 
? Tubes of bouillon or sterile water may be substituted 
for tubes Nos. 1 and 2, in which case, of course, but one 
plate can be made—z. ¢., from tube No. 3. 
6 
