252 LABORATORY EXERCISES IN BACTERIOLOGY. 



3. Kleb's Fractional or Dilution Method. This is a modification of the ordinary 

 plating methods introduced for use in case the original material is believed to contain 

 a large number of organisms, and where it is feared the degree of bacterial presence 

 is so great that a plate made from the medium inoculated with the undiluted substance 

 will be hopelessly crowded with colonies. It is essentially the same method of frac- 

 tional or dilution inoculation described in connection with the instruction for the 

 counting of bacteria in any material to be analyzed (page 162). It is very 

 popular, being perhaps more than any of the other mechanical methods employed 

 in isolation work. In this practice it is hoped by serial dilutions of the original 

 material to diminish the number and thoroughly disseminate the different types 

 of bacteria present so as to obtain in a final inoculation growth of but a single 

 variety or at best of but a few types. These dilutions of the infected material may 

 be made either in sterile water before inoculation, or by diffusions or smears in the 

 medium of growth in the course of inoculation. The latter is the method ordinarily 

 employed. For example, in the isolation of the mycobacterium of diphtheria from 

 the material obtained from the throat of a patient, a tube of liquefied blood-serum 

 is first inoculated by rubbing the swab on which the exudate has been collected upon 

 the surface of the medium. The swab is now returned to its protective tube and the 

 platinum loop taken up. This, having been sterilized in the flame and cooled, is 

 drawn over the surface of the serum in this first tube and then carried over the surface 

 of sterile serum in a second tube. It is again flamed and cooled and drawn over the 

 inoculated serum of the second tube, and the infection adhering to it carried to sterile 

 serum in a third tube. It is to be supposed that in these transferences but a small 

 number of the original organisms have found their way into the third tube, and these 

 few are probably widely scattered over the surface of the serum. Each tube is marked 

 in proper manner and placed in the incubator. The first growths to appear in the 

 medium, usually within eighteen or twenty hours, are the small white, punctate colonies 

 of Mycobacterium diphtheria. They are most definite and easily recognized in the 

 third tube ; and from this, as soon as discovered, should be transferred with the needle 

 to a fresh tube of serum, a stroke inoculation being made. (A film is also to be prepared 

 and stained with Loeffler's blue for confirmation.) Later, in all three tubes, espe- 

 cially in the first and second, appear in greater or less profusion the colonies of the 

 pus germs and other organisms, often to utter confusion. 



The same principle is followed where the dilution is made by diffusion in liquefied 

 gelatine or agar. A small quantity of the infected matter, as contaminated water, is 

 planted by diffusion in a tube of liquefied gelatine. From this tube a small quantity 

 (a loopful, or a definitely measured amount, representing a certain proportion of the 

 original quantity of the fluid examined, if study of the number of bacteria is also to 

 be pursued) is transferred to a second tube of liquefied gelatine and diffused ; from this 

 a similar quantity is transferred to a third tube and likewise diffused in the liquefied 

 medium in it. All three tubes are plated in one or other manner, and the plated 

 cultures placed in proper surroundings for development. In the last, from the di- 

 minished numbers of germs present, the colonies will probably be well separated from 

 each other, and transfer by means of the needle to fresh medium possible. The first 

 and perhaps the second cultures will probably be too crowded with growth to permit 

 advantageous manipulation, but they will at least serve as controls to establish whether 

 in the third culture all the types of bacteria in the original material have been 

 obtained. 



