84 ISOLATION OF AEROBIC MICRO-ORGANISMS 



1. The application of heat to the isolation of micro-organisms. 



Spore-forming organisms can resist temperatures of 80 to 100 C. and 

 even 105 C. for several minutes, but non-spore-bearing organisms are soon 

 destroyed when heated to about 60 C. Hence it will be easy to separate a 

 spore-bearing from a non-spore-bearing organism in a mixture containing 

 both ; it will only be necessary to heat the mixture for a few minutes to a 

 temperature between 80 and 105 C., according to the resistance of the 

 spore, and subsequently to sow it in a tube of broth. Thus a pure culture 

 of the anthrax bacillus can be obtained by heating an impure culture to 

 '80-85 C. for 5 minutes. 



An infusion of hay if heated to 100 C. for 10 minutes will give a pure 

 culture of the Bacillus sublilis. Similarly an infusion of potato chips incubated 

 for two or three days and then heated to 105 C. for 5 minutes will give 

 a pure culture of the potato bacillus, and so on. 



In carrying out the above experiments, it is necessary to work with fluid cultures 

 or suspensions, since organisms when dried or mixed with solid matter are much 

 more resistant to heat. It is further essential to the success of the method that all 

 parts of the culture fluid be raised to the required temperature, otherwise some 

 of the non-sporing forms will escape destruction and the experiment will be only 

 a partial success. 



The technique is as follows : 



1. Prepare a very fine Pasteur pipette with a constriction below the 

 wool (p. 75). 



2. Fill the pipette with the culture up to the constriction and seal both 

 ends in the flame. 



3. Immerse the tube in a water bath heated to the temperature required, 

 and leave it for 5 or 10 minutes. If the temperature required be above 

 100 C. the tube must be heated in the autoclave. 



4. Dry the tube and then break off one end with a pair of sterile forceps 

 after passing it through the flame. Withdraw a little of the fluid into another 

 sterile pipette, being careful to avoid contaminating it, and sow sub-cultures. 



2. Isolation by cultivation at the optimum temperature. 



Fractional cultivation. 



While some organisms will grow at any temperature between 10 and 

 40 C., the limits of temperature within which growth takes place are in 

 the majority of cases much more restricted. Thus a large number of 

 saprophytes grow slowly and poorly above 30 C. ; many of the pathogenic 

 bacteria attain their maximum development between 30 and 40 C., others 

 will not grow below 30 C., while yet another group (the typhoid-colon group) 

 grows at 43 C. a temperature which is too high for the multiplication of 

 most micro-organisms. These facts with regard to differences in the optimum 

 temperature at which micro-organisms grow are applied for the purpose of 

 isolating organisms in pure culture. 



For example, the colon bacillus can be isolated from stools by sowing a 

 trace of the material in broth and incubating at 43 C. Incubation at this 

 temperature however does not at once yield a pure culture, for the organisms 

 which were present with the colon bacillus in the original material have not 

 been destroyed but their growth merely arrested ; so that were a sub-culture 

 to be sown from this first broth culture and incubated at 37 C. these co- 

 existing organisms would multiply under the more favourable conditions and 

 contaminate the culture of the colon bacillus. To eliminate them the method 

 of fractional cultivation may conveniently be adopted ; thus when the first 



