252 METHODS OF ISOLATION 



late tube No. 2, by making three parallel streaks from 

 end to end of the slanted surface. 



5. Plant the remainder of the tubes in the series as 

 " smears" like tube No. i. 



6. Label with distinctive name or number, and date; 

 incubate. 



The growth that ensues in the first two or three 

 tubes of the series will probably be so crowded as to be 

 useless. Toward the end of the series, however, dis- 

 crete colonies will be found, each of which can be 

 transferred to a fresh tube of nutrient medium without 

 risk of contamination from the neighbouring colonies. 



"Working" up Plates. 



Having succeeded in obtaining a plate (or tube 

 cultivation) in which the colonies are well grown and 

 sufficiently separated from each other, the process of 

 " working up," " pricking out," or " fishing " the colonies 

 in order to obtain subcultures in a state of purity from 

 each of the different bacteria present must now be 

 proceeded with. 



Occasionally it happens that this is quite a simple 

 matter. For example, the original mixed cultivation 

 when examined microscopically was found to contain a 

 Gram positive micrococcus, a Gram positive straight 

 bacillus and a Gram negative short bacillus. The third 

 gelatine plate prepared from this mixture, on inspection 

 after four day's incubation, showed twenty-five colonies 

 seven moist yellow colonies, each sinking into a shal- 

 low pit of liquefied gelatine, fourteen flat irridescent 

 filmy colonies, and four raised white slimy colonies. 

 A film preparation (stained Gram) from each variety ex- 

 amined microscopically showed that the yellow liquefy- 

 ing colony was composed of Gram positive micrococci ; 

 the flat colony of Gram positive bacilli and the white 

 colony of gram negative bacilli. One of each of these 

 varieties of colonies would be transferred by means of 



