Cultivation and Identification of Bacteria 119 



where, it must be expected that they will occur as mixed cultures. 

 A number of technics designed to separate bacteria from their 

 neighbors are available, but before much can be done in the way of 

 identifying these organisms, it is necessary that the culture be pure. 



BROTH DILUTION 



This method represents one of the earliest attempts to secure 

 pure cultures of organisms. The first in a series of broth tubes is 

 inoculated with the material containing the bacteria, and after 

 thorough mixing, a small quantity ( a loopf ul or a drop ) from tube 

 4^1 is transferred to tube #2. After mixing the contents of tube 

 #2, a transfer is carried from #2 to tube #3, etc., in series. A 

 decreasing number of organisms is carried over into each succeed- 

 ing tube of broth, and if this dilution procedure is carried along 

 through a sufficient number of transfers, the point is eventually 

 reached where the inoculum consists of only one or a very few 

 cells. The species predominating in the original material logically 

 would be found in pure culture in the last tube of the series show- 

 ing visible growth after a suitable incubation period. 



A little contemplation should make it obvious that this broth 

 dilution technic has several serious drawbacks. First of all, one 

 can never be sure that the last dilution tube will always contain a 

 pure culture. If two species of organisms are found in about equal 

 numbers in the original test material, they might both be carried 

 over into the last dilution tube and yield a mixed culture. But a 

 serious disadvantage of the method is that it denies the opportunity 

 for isolating those species which happen to be in the minority in 

 the original microbial mixture. 



AGAR DILUTION 

 Bv incorporating a solidifying agent in the broth to be em- 

 ployed in the serial dilutions, it is possible to anchor the organisms 

 in the solid medium. If the melted agar is cooled to between 

 45-50° C. before inoculation and is poured into a culture dish be- 

 fore the medium thickens, the poured agar will solidify when the 

 temperature approaches 40° C, and the organisms are trapped 



