BIOLOGICAL METHODS 85 



broth culture incubated at 43 C. has become cloudy a trace of it is sown in 

 another tube of broth which is then similarly incubated at 43 C., and from 

 the second tube a third is sown, and so on, until after several sub-cultures 

 in series, each incubated at 43 C., a pure culture of the colon bacillus is 

 ultimately obtained. 



A method analogous to this is employed when it is required to isolate the cholera 

 vibrio from specifically infected stools, only in this particular case the action of 

 temperature (37-38 C.) is combined with that of a special medium (vide infra) in 

 which fractional cultivation is effected. This will be found to be in most cases quite 

 a useful method for eliminating saprophytic organisms. 



3. Isolation by cultivation on special media. 



The growth of any given organism to the exclusion of that of others which 

 are present with it may be effected by sowing the material on a medium 

 which is designed to meet the requirements of the organism to be isolated. 



The diphtheria bacillus, for instance, can be isolated in pure culture by 

 smearing the surface of a number of serum tubes with a piece of membrane. 

 Isolation in this case is favoured by the fact that serum is very well adapted 

 to the growth of the diphtheria bacillus, but more or less unfavourable to the 

 multiplication of organisms which are generally found associated with this 

 bacillus. 



For the isolation of the cholera vibrio, Koch and Metchnikoff recommend 

 special media which though of poor nutritive value happen to meet its 

 particular requirements. Thus a trace of the " rice water " stool is sown 

 in a tube of Metchnikoff's liquid peptone-gelatin medium (p. 33) and incu- 

 bated at 38 C. Under these circumstances the growth of the cholera vibrio 

 is much more rapid than that of the other organisms present. The vibrio 

 being a very strictly aerobic organism forms a pellicle on the surface of the 

 liquid, and if after the culture has been incubating for 12 hours or so, 

 a trace of the film be examined, it will be found to consist of an almost pure 

 culture of the cholera vibrio. To further purify the culture recourse must 

 be had to fractional cultivation [sowing the sub-culture with a trace of the 

 pellicle taken up on the point of a fine wire], and three passages will be all 

 that is necessary before finally plating out on gelatin as described on p. 78. 



[For the isolation of bacilli of the typhoid-colon group MacConkey has 

 introduced bile-salt media. The material suspected to contain the organism 

 is sown in a liquid bile-salt medium, and after incubation, preferably at 

 42 C., a trace of the culture is plated out on a bile-salt-agar and suspicious 

 colonies picked off for further examination (for fuller details of the method, 

 see Chaps. XXI. and XXIII.).] 



Finally, in some cases, the growth of associated organisms may be arrested 

 by adding to the medium some antiseptic which is not injurious to the 

 organism to be isolated. Chantemesse for instance advises the use of media 

 containing carbolic when attempting the isolation of the colon or typhoid 

 bacillus, and Eisner suggests the use of potassium-iodide-gelatin for the 

 same purpose. 



As has been indicated above, this and the method of cultivation at the 

 optimum temperature may be combined; Vincent for instance adopted a 

 combination of the two methods in his attempts to isolate the typhoid 

 bacillus (Chap. XXI.). 



4. Isolation by animal inoculation. 



In some cases the simplest, and perhaps the only, method of isolating a 

 pathogenic organism in pure culture from material in which it is mixed with 



