174 BIOLOGY AND TECHNIQUE 



THE ISOLATION OF BACTERIA IN PURE CULTURE 



It is obvious that in many cases where bacteria are cultivated 

 Irom water, milk, pathological material, or other sources, many 

 species may be present in the same specimen. It is likewise obvious 

 that scientific bacteriological study of any bacterium can be made 

 only if we obtain this particular species entirely apart from others, 

 in what is known as "pure culture." The earliest methods for 

 accomplishing this were the methods of Pasteur and of Cohn who 

 depended upon the power of one species to outgrow all others, if 

 cultivated for a sufficient length of time in fluid media. This method, 

 of course, was inadequate in that it was often purely a matter of 

 chance which one of the mixture of species was finally obtained by 

 itself. A later method, by Klebs, depends upon serial dilution, in 

 test tubes of fluid media, by which the eventual transference of 

 one germ only, to the last tube was attempted. Such methods, none 

 of them of great practical value, have been entirely displaced by 

 those made possible by Koch's introduction of solid media which 

 may be rendered fluid by heat. 



The methods now employed for the isolation of bacteria depend 

 upon the inoculation of gelatin or agar, when in the melted state, 

 the thorough distribution of the bacteria in these liquids by mixing, 

 and the subsequent congealing of these media in thin layers. By 

 this means the individual bacteria, distributed in the medium when 

 liquid, are held apart and separate when the medium becomes stiff. 

 The masses of growth or "colonies" which develop from these single 

 isolated microorganisms are discrete and are descendants of a single 

 organism, and can be transferred, by means of a process known 

 as "colony-fishing," to fresh sterile culture media. 



Plating. The first method employed by Koch for bacterial isola- 

 tions was one that consisted in the use of simple plates of glass, 

 about 3X4 inches in size, mounted upon a leveling stand. A 

 wooden triangle, supported upon three adjustable screw-feet, formed 

 the base of this apparatus. Upon this was set a covered crystallizing 

 dish which could be filled with ice water. Upon the top of this 

 rested the sterilized plates under a bell jar. By screwing up or 

 down upon the supports of the triangle, leveling of the plate could 

 be achieved and controlled by a spirit-level placed at its side. The 

 inoculated gelatin was poured upon the plate and spread and rapidly 



