PART II 



SYSTEMATIC BACTERIOLOGY 



CHAPTER 12 



THE METHODS OF OBTAINING PURE CULTURES, AND THE 

 IDENTIFICATION OF BACTERIA 



Methods op Obtaining Pure Cultures of Bacteria 



One of the first essentials in the study of bacteriology is the preparation and 

 maintenance of pure cultures of bacteria. Neglect of this leads inevitably to 

 confusion. During the sixties and seventies of last century, micro-organisms were 

 perforce cultivated in liquid media ; and as the preparation of pure cultures in 

 such media is often difficult and sometimes impossible relatively little progress 

 in the identification of particular species was made in these years. It was the 

 introduction of solid media by Robert Koch in 1881 that rendered possible the 

 easy separation of different organisms from one another in a mixed culture, and 

 provided a means for distinguishing macroscopically between different species of 

 bacteria. Koch found that on a suitable solid medium, such as potato or gelatin, 

 most organisms formed characteristic colonies by which they could be readily 

 identified ; by inoculation of separate colonies into tubes of a liquid medium, 

 pure cultures could be obtained. These could again be streaked on to a solid 

 medium, and if the resulting colonies were all of the same appearance, it might 

 be concluded that the liquid culture probably contained only one species of 

 bacterium. This method — Koch's plating method — affords the simplest and most 

 rapid means of separating one organism from another ; we shall later describe it 

 more fully. 



We have now at our command numerous methods of purifying cultures. Some 

 are of limited utility, or are suited solely to certain organisms ; others are of 

 wider applicability. Without discussing the technical details we shall give a 

 brief description of the principles underlying the more important of these 

 methods. 



A. Dilution Method. — In point of time, this was the first method introduced 

 for obtaining pure cultures of a bacterium, a method which we owe to Lister (1878). 

 The mixed culture is diluted with sterile tap water, or other suitable fluid, till there 

 is only about one organism in every two drops of the mixture. A series of tubes 

 containing broth is then seeded, each with one drop of the diluted culture. If the 

 dilution has been correctly gauged, there should be a growth in approximately 



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