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 for further use. To intro- 

 duce a culture 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 is in 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 011 the kitchen 

 stove. 1 



To inoculate the liquid contained in one of these little flasks with mi- 

 croorganisms 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 microorganisms are associated which have a different ther- 



1 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. 



