CULTURES IN LIQUID MEDIA. 67 
is hermetically sealed. Thus one little flask after another is made from the 
same piece of tubing until this becomes too short forfurther use. To intro- 
duce aculture liquid into one of these little flasks, heat the bulb slightly, 
break off the sealed extremity of the tube and plunge it beneath the surface 
of the liquid (Fig. 37). The quantity which enters will of course depend 
upon the heat employed and the consequent rarefaction of the enclosed air. 
Ordinarily the bulb is filled to about one-third of its capacity with the cul- 
ture liquid, leaving it two-thirds full of air for the use of the microscopic 
plants which are to be cultivated in it. . . . Sterilization is effected by heat 
after the liquid has been introduced and the neck of the flask hermetically 
sealed in the flame of an alcohol lamp. —_— 
Sterilization may be effected by boiling for an hour in a bath of paraffin 
or of concentrated salt solution, by which a temperature considerably above 
that of boiling water is secured. The writer isin the habit of preparing a 
considerable number of these flasks at one time, and leaving them, in a suit- 
able vessel filled with water, for twenty-four hours or longer on the kitchen 
stove.! 
To inoculate the liquid contained in one of these little flasks with mi- 
cro6rganisms from any source, the end of the tube is first heated to destroy 
germs attached to the exterior; the extremity is then broken off with steril- 
ized (by heat) forceps; the bulb is very gently heated, so as to force out a 
little air, and the open end is plunged into the liquid containing the organ- 
ism to be cultivated (or into a vein, or one of the solid viscera of an animal 
dead from an infectious germ disease, such as anthrax). 
Inoculation from one tube to another may also be effected by means of 
the ordinary platinum wire needle. 
Before the introduction of Koch’s plate method for isolating bac- 
teria in pure cultures, certain methods had been proposed, and em- 
ployed to some extent, which at present have a historical value only. 
Thus Klebs (1873) proposed to take from a first culture in which 
two or more species were associated a minute quantity, by means of a 
capillary tube, and with this to inoculate a second culture. By re- 
peating this procedure several times he expected to exclude all except 
the species which was present in the greatest abundance and which 
multiplied most rapidly in the medium employed. 
The method by dilution, first employed with precision by Brefeld 
(1872) in obtaining pure cultures of mould fungi, and subsequently 
by Lister for the isolation of bacteria, consists in so diluting a minute 
quantity of the mixed culture that the number of bacteria in the dilu- 
tion may be less than one for each drop of the liquid. If now a 
single drop be added to each of a series of tubes containing a small 
quantity of sterile bouillon, some of the inoculations made may give 
a pure culture, as the drop may have contained but a single vege- 
tative cell. , 
Another method of obtaining a pure culture in liquid media, when 
several microdrganisms are associated which have a different ther- 
" Where a steam sterilizer is at hand they will be most conveniently sterilized in 
the usual way, by subjecting them to the boiling temperature for an hour at a time 
on three successive days. 
